OP-3- induced morphongenesis

ABSTRACT

Disclosed are (1) nucleic acid and amino acid sequences for a novel morphogenic protein; (2) methods for producing and expressing the protein in a biologically active form; and (3) methods for utilizing the protein to induce tissue morphogenesis in a mammal, including methods for increasing a progenitor cell population in a mammal, methods for stimulating progenitor cells to differentiate and maintain their differentiated phenotype in vivo or in vitro, methods for inducing tissue-specific growth in vivo and methods for the replacement of diseased or damaged tissue in vivo.

REFERENCE TO RELATED APPLICATIONS

This application is a divisional of copending U.S. application Ser. No.08/479,666, filed on Jun. 7, 1995, U.S. Pat. No. 5,652,337, which is adivisional of U.S. Ser. No. 07/971,091, filed Nov. 3, 1992, abandoned,which is a continuation-in-part of: (1) U.S. Ser. No. 07/922,813, filedJul. 31, 1992, abandoned, which is a continuation-in-part of U.S. Ser.No. 07/752,964, filed Aug. 29, 1991, abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/667,274, filed Mar. 11, 1991abandoned; (2) U.S. Ser. No. 07/923,780, filed Jul. 31, 1992, abandoned,which is a continuation-in-part of U.S. Ser. No. 07/752,764 and U.S.Ser. No. 07/752,857, both filed Aug. 30, 1991, both abandoned, and bothcontinuations-in-part of U.S. Ser. No. 07/667,274, filed Mar. 11, 1991abandoned,; (3) U.S. Ser. No. 07/938,336 and U.S. Ser. No. 07/938,337,both filed Aug.28, 1992, both abandoned, and both continuations-in-partof U.S. Ser. No. 07/753,059, filed Aug. 30, 1991, abandoned, which is acontinuation-in-part of U.S. Ser. No. 07/667,274, filed Mar. 11, 1991abandoned; (4) U.S. Ser. No. 07/938,021, filed Aug. 28, 1992, abandoned,which is a continuation-in-part of U.S. Ser. No. 07/752,861, filed Aug.30, 1991, abandoned, which is a continuation-in-part of U.S. Ser. No.07/667,274, filed Mar. 11, 1991 abandoned; (5) U.S. Ser. No. 07/945,285and U.S. Ser. No. 07/945,286, both filed on Sep. 15, 1992, bothabandoned, and both continuations-in-part of U.S. Ser. No. 07/752,764,filed Aug. 30, 1991, abandoned, which is a continuation-in-part of U.S.Ser. No. 07/667,274, filed Mar. 11, 1991 abandoned; (6) U.S. Ser. No.07/946,235 and U.S. Ser. No. 07/946,238, both filed Sep. 16, 1992, bothabandoned, and both continuations-in-part of U.S. Ser. No. 07/752,764,filed Aug. 30, 1991, abandoned, which is a continuation-in-part of U.S.Ser. No. 07/667,274, filed Mar. 11, 1991 abandoned. The disclosures ofthese applications are incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates generally to the field of tissue morphogenesisand more particularly to a novel protein that induces tissuemorphogenesis in mammals.

BACKGROUND OF THE INVENTION

Cell differentiation is the central characteristic of morphogenesiswhich initiates in the embryo, and continues to various degreesthroughout the life of an organism in adult tissue repair andregeneration mechanisms. The degree of morphogenesis in adult tissuevaries among different tissues and is related, among other things, tothe degree of cell turnover in a given tissue. On this basis, tissuescan be divided into three broad categories: (1) tissues with static cellpopulations such as nerve and skeletal muscle where there is no celldivision and most of the cells formed during early development persistthroughout adult life; (2) tissues containing conditionally renewingpopulations such as liver where there is generally little cell divisionbut, in response to an appropriate stimulus, cells can divide to producedaughters of the same differentially defined type; and (3) tissues withpermanently renewing populations including blood, testes and stratifiedsquamous epithelia which are characterized by rapid and continuous cellturnover in the adult. Here, the terminally differentiated cells have arelatively short life span and are replaced through proliferation of adistinct subpopulation of cells, known as stem or progenitor cells.

The cellular and molecular events which govern the stimulus fordifferentiation of these cells is an area of intensive research. In themedical field, it is anticipated that the discovery of factor(s) whichcontrol cell differentiation and tissue morphogenesis will advancesignificantly medicine's ability to repair and regenerate diseased ordamaged mammalian tissues and organs. Particularly useful areas includereconstructive surgery and in the treatment of tissue degenerativediseases including arthritis, emphysema, osteoporosis, cardiomyopathy,cirrhosis, and degenerative nerve diseases.

A number of different factors have been isolated in recent years whichappear to play a role in cell differentiation. Recently, various membersof the structurally related proteins of the transforming growth factor(TGF)-β superfamily of proteins have been identified as true morphogens.

This "family" of proteins, sharing substantial amino acid sequencehomology within their morphogenically active C-terminal domains,including a conserved six or seven cysteine skeleton, are capable ofinducing tissue-specific morphogenesis in a variety of organs andtissues, including bone, cartilage, liver, dentin, periodontal ligament,cementum, nerve tissue and the epithelial mucosa of the gastrointestinaltract. The proteins apparently bind to surface receptors or otherwisecontact and interact with progenitor cells, predisposing or stimulatingthe cells to proliferate and differentiate in a morphogenicallypermissive environment. The morphogens are capable of inducing thedevelopmental cascade of cellular and molecular events that culminate inthe formation of new organ-specific tissue, including anyvascularization, connective tissue formation, and nerve ennervation asrequired by the naturally occurring tissue.

Among the proteins useful in tissue morphogenesis are proteinsoriginally identified as bone inductive proteins, such as the OP-1,(also referred to in related applications as "OP1"), OP-2 (also referredto in related applications as "OP2"), and the CBMP2 proteins, as well asamino acid sequence-related proteins such as BMP5, BMP6 and its murinehomolog, Vgr-1, DPP and 60A (from Drosophila), Vg1 (from Xenopus), andGDF-1 (from mouse see, for example, U.S. Ser. No. 752,764, abandoned,U.S. Ser. No. 667,274, abandoned, and U.S. Ser. No. 923,780, abandoned,and PCT documents US92/01968 and US92/07358). These TGF-β superfamilymembers comprise a distinct subfamily of proteins different from othermembers of the TGF-β superfamily in that the family of morphogenicproteins are able to induce the full cascade of events that result intissue morphogenesis, including stimulating cell proliferation and celldifferentiation, supporting the growth and maintenance of differentiatedcells and inducing the "redifferentiation" of transformed cells todisplay a morphology characteristic of untransformed cells. Themorphogenic proteins apparently can act as endocrine, paracrine orautocrine factors. Specifically, the endogenous morphogens may besynthesized by the cells on which they act, by neighboring cells, or bycells of a distant tissue, the secreted protein being transported to thecells to be acted on. In addition, the family of morphogenic proteinsinduce true tissue morphogenesis, rather than inducing formation offibrotic (scar) tissue as, for example, TGF-β does.

The morphogens are synthesized in the cell as a precursor moleculeapproximately three times larger than the mature protein that isprocessed to yield mature disulfide-linked dimers comprising theC-terminal domain of the precursor sequence. The proteins are inactivewhen reduced e.g., in monomeric form, but are active as oxidizedhomodimeric species as well as when oxidized in combination with othermorphogens to produce heterodimers. The proteins useful in tissuemorphogenesis typically require a suitable environment enabling cells tomigrate, proliferate and differentiate in a tissue-specific manner into,e.g., cartilage-producing chondroblasts, bone-producing osteoblasts,hemopoietic cells, or liver cells, depending on the nature of the localenvironment. The proliferation and differentiation of cells induced bythe morphogenic proteins requires a suitable local environment,including a suitable substratum on which the cells can anchor. Theproliferating and differentiating cells also require the presence ofappropriate signals to direct their tissue-specificity, such as cellsurface markers.

It is an object of this invention to provide a novel purifiedmorphogenic protein, "OP-3", including the amino acid sequence definingit and nucleic acids encoding it, including allelic, species, mutant andchimeric variants thereof, and methods for utilizing the protein toinduce the developmental cascade of tissue morphogenesis for a varietyof tissues in mammals. The morphogenic properties of OP-3 include theability to induce proliferation and differentiation of progenitor cells,and the ability to support and maintain the differentiated phenotypethrough the progression of events that results in the formation of adulttissue. Another object is to provide methods for the expression andisolation of morphogenically active species of OP-3 using recombinantDNA techniques. Yet another object is to provide generic sequencesdefining useful morphogens. Still another object is to providetissue-specific acellular matrices that may be used in combination withOP-3, and methods for their preparation. Other objects include utilizingOP-3 in a variety of applications including methods for increasing aprogenitor cell population in a mammal; methods for stimulatingprogenitor cells to differentiate in vivo or in vitro and to maintaintheir differentiated phenotype; methods for inducing tissue-specificgrowth in vivo, and methods for the replacement of diseased or damagedtissue in vivo. These and other objects and features of the inventionwill be apparent from the description, drawings, and claims whichfollow.

SUMMARY OF THE INVENTION

A novel substantially pure genetic sequence encoding a novelsubstantially pure protein referred to herein as "OP-3" now has beendiscovered. This novel protein is a member of the morphogenic proteinfamily previously described by Applicants (see U.S. Ser. No. 667,274,abandoned and U.S. Ser. No. 752,764, abandoned). Accordingly, theinvention provides methods for utilizing OP-3 to induce thedevelopmental cascade of tissue morphogenesis in a mammal. Specifically,methods are provided for utilizing OP-3 to induce the proliferation ofuncommitted progenitor cells, to induce the differentiation of thesestimulated progenitor cells in a tissue-specific manner underappropriate environmental conditions, and to support the growth andmaintenance of these differentiated cells. The protein also may be usedto stimulate the "redifferentiation" of cells that have strayed fromtheir differentiated phenotypes. Accordingly, OP-3 can be utilized toinitiate and maintain the developmental cascade of tissue morphogenesisin an appropriate, morphogenically permissive environment.

As used herein, useful OP-3 morphogens include proteins encoded by theDNA sequence provided in Seq. ID No. 1 ("mOP-3") and allelic and speciesvariants thereof, as well as other naturally-occurring and biosyntheticmutants, including chimeric proteins, that are morphogenically active asdefined herein. "Morphogenically active fragment" is understood toinclude all proteins and protein fragments encoded by part or all of thesequence of Seq. ID No. 1 and which have morphogenic activity as definedherein. Specifically, as defined herein, a morphogen is a dimericprotein comprising a pair of polypeptide chains, wherein eachpolypeptide chain comprises at least the C-terminal six cysteineskeleton defined by residues 303 to 399 of Seq. ID No. 1 (or residues335-431 of OP1, Seq. ID no. 3), including functionally equivalentarrangements of these cysteines (e.g., amino acid insertions ordeletions which alter the linear arrangement of the cysteines in thesequence but not their relationship in the folded structure), such that,when the polypeptide chains are folded, the dimeric protein speciescomprising the pair of polypeptide chains has the appropriatethree-dimensional structure, including the appropriate intra- orinter-chain disulfide bonds such that the protein is capable of actingas a morphogen as defined herein. Specifically, the morphogens generallyare capable of all of the following biological functions in amorphogenically permissive environment: stimulating proliferation ofprogenitor cells; stimulating the differentiation of progenitor cells;stimulating the proliferation of differentiated cells; and supportingthe growth and maintenance of differentiated cells, including the"redifferentiation" of transformed cells. In addition, it is alsoanticipated that these morphogens are capable of inducingredifferentiation of committed cells under appropriate environmentalconditions.

In one aspect, the morphogens of this invention comprise amorphogenically active dimeric species comprising a pair of polypeptidechains, wherein at least one of the polypeptide chains comprises theamino acid sequence defined by residues 303 to 399 of Seq. ID No. 1including allelic, species and other mutant variants thereof. Inpreferred morphogens, at least one polypeptide chain comprises thesequence defined by residues 298-399, residues 261-399 or residues264-399 of Seq. ID No. 1. Alternatively, the amino acid sequence of bothpolypeptide chains may be defined by part or all of the amino acidsequence of Seq. ID No. 1, including allelic, species and other mutantvariants thereof including chimeric constructs as described below. Whereonly one polypeptide chain is defined by the amino acid sequence of partor all of Seq. ID. No. 1, the other polypeptide chain preferablycomprises at least the sequence defining the C-terminal six cysteineskeleton of any of the other known morphogen family members, includingOP-1, OP-2, CBMP2A, CBMP2B, BMP3, BMP5, BMP6, Vgr-1, Vg1, 60A, DPP andGDF-1 (described, for example, in U.S. Ser. Nos. 752,764, abandoned923,780, abandoned or US92/07358), including allelic, species and othermutant variants thereof, including chimeric variants. Other usefulsequences include biosynthetic constructs, such as are described in U.S.Pat. No. 5,011,691.

In still another aspect of the invention, generic sequences are providedwhich accommodate the sequence identity of useful morphogens andincorporate OP-3's novel features.

In another aspect of the invention, morphogens of this inventioncomprise morphogenically active proteins encoded by part or all of thegenetic sequence listed in Seq. ID No. 1, including allelic, species andother mutant variants thereof. In still another aspect, the inventioncomprises morphogens encoded by nucleic acids that hybridize to part orall of the pro region of the OP-3 protein, bases 120 to 848 of Seq IDNo. 1, under stringent hybridization conditions. As used herein,"stringent hybridization conditions" are defined as hybridization in 40%formamide, 5×SSPE, 5×Denhardt's Solution, and 0.1% SDS at 37° C.overnight, and washing in 0.1×SSPE, 0.1% SDS at 50° C..

In one aspect of the invention, morphogenically active fragments of OP-3are useful in the replacement of diseased or damaged tissue in a mammal,including, but not limited to, damaged lung tissue resulting fromemphysema; cirrhotic tissue, including cirrhotic kidney or liver tissue;damaged heart or blood vessel tissue, as may result fromcardiomyopathies and/or atherothrombotic or cardioembolic strokes;damaged stomach and other mucosal tissues of the gastrointestinal tractresulting from ulceric perforations and/or their repair; damaged nervetissue as may result from physical injury, degenerative diseases such asAlzheimer's disease, multiple sclerosis, or strokes; damaged cartilageand bone tissue as may result from metabolic bone diseases and otherbone remodeling disorders; damaged dentin, periodontal and/or cementumtissue as may result from disease or mechanical injury; and in thereplacement of damaged tissue as a result of inflammation and/or chronicinflammatory disease.

As provided herein, morphogenically active fragments of OP-3 areprovided to a tissue-specific locus in vivo, to induce the developmentalcascade of tissue morphogenesis at that site. Cells stimulated ex vivoby contact with OP-3 also may be provided to the tissue locus. In thesecases the existing tissue provides the necessary matrix requirements,providing a suitable substratum or scaffold for the proliferating anddifferentiating cells in a morphogenically permissive environment, aswell as providing the necessary signals for directing thetissue-specificity of the developing tissue. The proteins or stimulatedcells also may be combined with a formulated matrix and implanted as adevice at a locus in vivo. The formulated matrix should be abiocompatible, preferably biodegradable acellular matrix having thecharacteristics described below. Where the necessary signals fordirecting the tissue-specificity of the developing tissue are notprovided endogenously, the matrix preferably also is tissue-specific.

In another aspect, the members of the morphogen protein family also cancontrol the body's cellular and humoral inflammatory response to aforeign object or an initial tissue injury. In many instances, the lossof tissue function results from the tissue destructive effects and thesubsequent formation of scar tissue associated with the body'simmune/inflammatory response to an initial or repeated injury to thetissue. The degree of scar tissue formation generally depends on theregenerative properties of the injured tissue, and on the degree andtype of tissue damage. Thus; in another aspect, morphogenically activefragments of OP-3 may be used to prevent or to substantially inhibit theformation of scar tissue, including alleviating immune response-mediatedtissue damage, by providing OP-3 or cells stimulated by exposure to OP-3protein, to a newly injured tissue locus. The OP-3 protein also may beprovided as a prophylactic, provided to a site in anticipation of tissueinjury, such as part of a surgical or other clinical procedure likely toproduce tissue damage, and to induce an inflammatory/immune response. Ina particularly useful embodiment, OP-3 may be used as part of atransplant procedure, to enhance the tissue viability of the organand/or tissue to be transplanted. The morphogen may be provided to theorgan and/or tissue to be transplanted prior to harvest, during itstransport, and/or during transplantation into the recipient host asdescribed below.

OP-3 also may be used to increase or regenerate a mesenchymal progenitoror stem cell population in a mammal. For example, progenitor cells maybe isolated from an individual's bone marrow, stimulated ex vivo withmorphogenic OP-3 for a time and at a concentration sufficient to inducethe cells to proliferate, and returned to the bone marrow. Other sourcesof progenitor cells that may be suitable include biocompatible cellsobtained from a cultured cell line, stimulated in culture, andsubsequently provided to the body. Alternatively, OP-3 may be providedby systemic (e.g., oral or parenteral) administration, or it may beinjected or otherwise provided to a progenitor cell population in anindividual to induce its mitogenic activity in vivo. For example, amorphogenically active fragment of OP-3 may be provided to the cells invivo, e.g., by systemic injection, to induce mitogenic activity.Similarly, a particular population of hemopoietic stem cells may beincreased by exposure to OP-3, for example by perfusing(plasmaphoresing) an individual's blood to extract the cells ofinterest, stimulating these cells ex vivo, and returning the stimulatedcells to the blood.

It is anticipated that the ability to augment an individual's progenitorcell population will enhance existing methods for treating disordersresulting from a loss or reduction of a renewable cell populationsignificantly. Two particularly significant applications include thetreatment of blood disorders and diseases involving impaired or lostimmune function.

The morphogens of this invention also can inhibit proliferation ofepithelial cell populations. The ability to inhibit epithelial cellproliferation may be exploited to reduce tissue damage associated withpsoriasis and dermatitis, and other inflammatory skin diseases, as wellas ulcerative diseases of the gastrointestinal tract, such as, forexample, in the healing of ulcers, including gastric ulcers, and theulcerations induced in oral mucocitis and inflammatory bowel disease.Morphogens may be used to particular advantage as a cytoprotective agentin clinical therapies likely to effect proliferating epithelialpopulations, such as cancer radiotherapies and chemotherapies thattypically induce oral mucositis, hair loss and/or skin disorders.

In another aspect of the invention, morphogenic OP-3 may be used tosupport the growth and maintenance of differentiated cells, inducingexisting differentiated cells to continue expressing their phenotype. Itis anticipated that this activity will be particularly useful in thetreatment of tissue disorders where loss of function is caused byreduced or lost metabolic function in which cells become senescent orquiescent, such as may occur in aging cells and/or may be manifested inosteoporosis and a number of nerve degenerative diseases, includingAlzheimer's disease. Application of OP-3 directly to the cells to betreated, or providing it systemically, as by oral or parenteraladministration, can stimulate these cells to continue expressing theirphenotype, thereby significantly reversing the effects of thedysfunction. In addition, a morphogenically active fragment of OP-3 alsomay be used in gene therapy protocols to stimulate the growth ofquiescent cells, thereby potentially enhancing the ability of thesecells to incorporate exogenous DNA.

In yet another aspect of the invention, a morphogenically activefragment of OP-3 also may be used to induce "redifferentiation" of cellsthat have strayed from their differentiation pathway, such as can occurduring tumorgenesis. It is anticipated that this activity will beparticularly useful in treatments to reduce or substantially inhibit thegrowth of neoplasms. The method also is anticipated to induce the de-and/or re-differentiation of these cells. As described supra, amorphogenically active OP-3 fragment may be provided to the cellsdirectly or systemically, stimulating these cells to revert back to amorphology and phenotype characteristic of untransformed cells.

In still another aspect of the invention, OP-3 may be used to stimulatecell adhesion molecule (CAM) expression levels in a cell. CAMs aremolecules defined as carrying out cell-cell interactions necessary fortissue formation. CAMs are believed to play a fundamental regulatoryrole in tissue development, including tissue boundary formation,embryonic induction and migration, and tissue stabilization andregeneration. Altered CAM levels have been implicated in a number oftissue disorders, including congenital defects, neoplasias, anddegenerative diseases.

In particular, N-CAM expression is associated with normal neuronal celldevelopment and differentiation, including retinal formation,synaptogenesis, and nerve-muscle tissue adhesion. Inhibition of one ormore of the N-CAM isoforms is known to prevent proper tissuedevelopment. Altered N-CAM expression levels also are associated withneoplasias, including neuroblastomas (see infra), as well as with anumber of neuropathies, including normal pressure hydrocephalous andtype II schizophrenia. Application of the morphogen directly to thecells to be treated, or providing the morphogen to the mammalsystemically, for example, parenterally, or indirectly by oraladministration, may be used to induce cellular expression of one or moreCAMs, particularly N-CAMs and L1.

CAMs also have been postulated as part of a morphoregulatory pathwaywhose activity is induced by a to date unidentified molecule (See, forexample, Edelman, G. M. (1986) Ann. Rev. Cell Biol., 2:81-116). Withoutbeing limited to any given theory, the morphogens described herein mayact as inducers of this pathway.

The matrices utilized in the methods of the invention may be derivedfrom organ-specific tissue, or they may be formulated synthetically. Inone embodiment of the invention, when OP-3 (or a collection ofprogenitor cells stimulated by OP-3) is provided at a tissue-specificlocus, e.g., by systemic administration, implantation or injection at atissue-specific locus, the existing tissue at that locus, whetherdiseased or damaged, has the capacity of acting as a suitable matrix orscaffold for the differentiation and proliferation of migratingprogenitor cells. Alternatively, a formulated matrix may be providedexternally together with the stimulated progenitor cells ormorphogenically active OP-3 fragment, as may be necessary when theextent of injury sustained by the damaged tissue is large. The matrixshould be a biocompatible, suitably modified acellular matrix havingdimensions such that it allows the differentiation and proliferation ofmigratory progenitor cells, and is capable of providing amorphogenically permissive environment. The matrix also preferablyallows cellular attachment and is biodegradable. Where the necessarytissue-directing signals can not be provided endogenously, the matrixpreferably also is tissue-specific.

Formulated matrices may be generated from dehydrated organ-specifictissue prepared, for example, by treating the tissue with solvents tosubstantially remove the intracellular, non-structural components fromthe tissue. Alternatively, the matrix may be formulated syntheticallyusing a biocompatible, preferably in vivo biodegradable, structuralmolecule, and may be formulated with suitable tissue-specific cellattachment factors. The molecule may be a naturally occurring one suchas collagen, laminin or hyaluronic acid, or a synthetic polymercomprising, for example, polylactic acid, polybutyric acid, polyglycolicacid, and copolymers thereof. Currently preferred structural polymerscomprise tissue-specific collagens. Currently preferred cell attachmentfactors include glycosaminoglycans and proteoglycans. The matrix furthermay be treated with an agent or agents to increase the number of poresand micropits on its surfaces, so as to enhance the influx,proliferation and differentiation of migratory progenitor cells from thebody of the mammal.

The invention thus relates to compositions and methods for the use ofmorphogenically active fragments of OP-3, a novel species variant of thegeneric family of morphogens disclosed in U.S. Ser. No. 667,274,abandoned and U.S. Ser. No. 752,764, abandoned, as a tissue morphogen.Morphogenically active OP-3 and protein fragments can be isolated fromnaturally-occurring sources, or they may be constructed biosyntheticallyusing conventional recombinant DNA technology. Active OP-3 useful in thecompositions and methods of this invention may include forms havingvarying glycosylation patterns, varying N-termini and active truncatedforms, e.g., produced by recombinant DNA techniques. Active OP-3proteins also include chimeric constructs as described below, comprisingboth an OP-3 active domain and a non-OP-3 sequence as, for example, thepro domain and/or the N-terminal region of the mature protein. OP-3protein can be expressed from intact or truncated cDNA or from syntheticDNAs in procaryotic or eucaryotic host cells, and purified, cleaved,refolded, and dimerized to form morphogenically active compositions.Useful host cells include procaryotes, including E. coli, and eucaryoticcells, including mammalian cells, such as CHO, COS, melanoma or BSCcells, or the insect/baculovirus system. Thus recombinant DNA techniquesmay be utilized to produce large quantities of OP-3 capable of inducingtissue-specific cell differentiation and tissue morphogenesis in avariety of mammals, including humans.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a nucleotide sequence comparison of the mouse cDNA sequence ofOP-2 (bases 93-1289 of SEQ ID NO:9) and OP-3 (bases 69-1265 of SEQ IDNO:1). Exon boundaries are indicated by bars beneath the sequence;diamonds indicate nucleotide differences within exons 2 and 3.

DETAILED DESCRIPTION

The invention provides a novel genetic sequence, mOP-3, encoding a novelprotein, OP-3, having morphogenic properties. The genetic sequenceoriginally was identified in a mouse cDNA library, and the inventionprovides methods for identifying and isolating the gene from otherspecies. As will be appreciated by those skilled in the art, the methodsdescribed herein also may be used to isolate the OP-3 gene from otherlibraries, including genomic libraries. The invention also providesmeans for producing the OP-3 genetic sequence and the encoded protein.The invention further provides methods and compositions for inducing thedevelopmental cascade of tissue morphogenesis in a mammal utilizingmorphogenically active fragments of OP-3. The methods and compositions.provided herein may be utilized in a range of applications, includingstimulating the proliferation and/or differentiation of progenitor cellsand inducing the repair and regeneration of damaged tissue. Themorphogenic OP-3 species of the invention are novel species variants ofthe family of morphogens disclosed in copending U.S. Ser. No. 667,274,U.S. Ser. No. 752,764, U.S. Ser. No. 923,780 and U.S. Ser. No. 922,813,all abandoned, the disclosures of which are incorporated hereinabove byreference. As described herein, OP-3 may be isolated from naturalsources or constructed biosynthetically utilizing conventionalrecombinant DNA technology or constructed synthetically using standardchemical techniques.

Morphogenically active fragments of OP-3 are useful for initiating andmaintaining the tissue-specific developmental cascade in a variety oftissues, including, but not limited to, bone, cartilage, dentin, neuraltissue, liver, periodontal ligament, cementum, lung, heart, kidney andnumerous tissues of the gastrointestinal tract. When combined with naivemesenchymal progenitor cells as disclosed herein, OP-3 can induce theproliferation and differentiation of these progenitor cells. In thepresence of appropriate tissue-specific signals to direct thedifferentiation of these cells, and a morphogenically permissiveenvironment, OP-3 is capable of reproducing the cascade of cellular andmolecular events that occur during embryonic development to yieldfunctional tissue. For example, the protein can induce the de novoformation of cartilage and endochondral bone, including inducing theproliferation and differentiation of progenitor cells into chondrocytesand osteoblasts, inducing appropriate mineralization and boneremodeling, inducing formation of an appropriate bone tissue vascularsupply and inducing formation of differentiated bone marrow (see Example7 below.)

Provided below is a detailed description of the nucleic acid and aminoacid sequences which describe OP-3 proteins useful in the compositionsand methods of this invention, including a description of how to makethem, and methods and means for their therapeutic administration. Alsoprovided are numerous, nonlimiting examples which (1) illustrate thesuitability of these proteins as tissue morphogens and therapeuticagents, and (2) provide assays with which to test the morphogensencompassed by the invention in different tissues. Also provided inExample 9 is a method for screening compounds to identify morphogenstimulating agents capable of stimulating endogenous OP-3 expressionand/or secretion. OP-3 stimulating agents then may be used in any of thetherapeutic applications described herein in place of, or in additionto, OP-3 protein administration.

I. Useful Morphogens

As defined herein a protein is morphogenic if it is capable of inducingthe developmental cascade of cellular and molecular events thatculminate in the formation of new, organ-specific tissue and comprisesat least the conserved C-terminal six cysteine skeleton or itsfunctional equivalent (see supra). Specifically, the morphogensgenerally are capable of all of the following biological functions in amorphogenically permissive environment: stimulating proliferation ofprogenitor cells; stimulating the differentiation of progenitor cells;stimulating the proliferation of differentiated cells; and supportingthe growth and maintenance of differentiated cells, including the"redifferentiation" of transformed cells. Details of how the morphogenfamily of proteins described herein first were identified, as well as adescription of how to make, use and test them for morphogenic activityare disclosed in U.S. Ser. No. 667,274, filed Mar. 11, 1991, abandoned,and U.S. Ser. No. 752,764, filed Aug. 30, 1991, abandoned. As disclosedtherein, the morphogens may be purified from naturally-sourced materialor recombinantly produced from procaryotic or eucaryotic host cells,preferably as described therein. Alternatively, novel morphogenicsequences may be identified following the procedures disclosed therein.

Particularly useful morphogens identified to date include OP-1, OP-2,CBMP2A and CBMP2B (the morphogenically active domains of proteinsreferred to in the art as BMP2A and BMP2B, or BMP2 and BMP4,respectively), BMP3, BMP5, BMP6, Vgr-1, GDF-1, Vg1, DPP and 60A,including their allelic and species variants, as well as other mutantvariants including chimeric morphogens. Detailed descriptions of theproteins may be found in, for example, U.S. Ser. No. 752,764, abandonedor U.S. Ser. No. 922,813, abandoned. Morphogenically active biosyntheticconstructs such as those disclosed in U.S. Pat. No. 5,011,691, thedisclosure of which is incorporated herein by reference (e.g., COP-1,COP-3, COP-4, COP-5, COP-7, and COP-16) also are envisioned to beuseful.

Using the methodology disclosed in U.S. Ser. No. 752,764, abandoned, andU.S. Ser. No. 667,274, abandoned, the novel morphogen OP-3 and itsgenetic sequence, now have been identified. The OP-3 proteins useful inthe invention include any morphogenically active fragment of the OP-3amino acid sequence present in Seq. ID No. 1, or allelic, species orother mutant variants thereof. The morphogenically active fragment ofOP-3 also may include any morphogenically active protein encoded by partor all of the nucleic acid sequence presented in Seq. ID No. 1. Themorphogenic protein also may comprise a protein encoded by part or allof a nucleic acid which hybridizes to at least part of the nucleic acidsequence encoding the "pro" region of the OP-3 protein, bases 120-848 ofSeq. ID No. 1, under stringent conditions.

The mOP-3 gene encodes a protein ("mOP-3") first expressed as animmature translation product that is 399 amino acids in length. Thisprecursor form, referred to herein as the "prepro" form, (Seq. ID. No.1, amino acid residues 1-399) includes an N-terminal signal peptidesequence, typically less than about 20 residues, followed by a "pro"domain that is cleaved to yield the mature sequence. The "pro" form ofthe protein includes the pro domain and the mature domain, and forms asoluble species that appears to be the primary form secreted fromcultured mammalian cells. The signal peptide is cleaved rapidly upontranslation, at a cleavage site that can be predicted in a givensequence using the method of Von Heijne ((1986) Nucleic Acids Research14:4683-4691). The preferred form of morphogenically active OP-3 proteincomprises a processed sequence, including fragments thereof,appropriately dimerized and disulfide bonded. Where a soluble form ofthe protein is desired, the protein preferably comprises both the maturedomain, or an active portion thereof, and part or all of the pro domain.

By amino acid sequence homology with other, known morphogens, the prodomain likely is cleaved at residues 257-260 of Seq. ID No. 1, whichrepresent the canonical Arg-Xaa-Xaa-Arg cleavage site, to yield a maturesequence 139 amino acids in length (Seq. ID No. 1, residues 261-399).Alternatively, the pro domain may be cleaved at residues 260-263 toyield a shorter sequence 135 amino acids in length (Seq. ID No. 1, aminoacid residues 264-399). All morphogens, including OP-1, OP-2 and theOP-3 proteins disclosed herein, comprise at least a conserved sixcysteine skeleton in the amino acid sequence C-terminal domain and,preferably, a conserved seven cysteine skeleton (see, for example, U.S.Ser. No. 752,764, abandoned). The conserved six cysteine skeleton inmOP-3 (Seq. ID No. 1) is defined by amino acid residues 303-399; theconserved seven cysteine skeleton is defined by amino acid residues298-399. In addition to the conserved six cysteine skeleton found inknown morphogen family members including OP-1, OP-2, CBMP2A, CBMP2B,BMP3, BMP5, BMP6, Vgr-1, Vg1, 60A, DPP and GDF-1, (described, forexample, in U.S. Ser. No. 752,764, abandoned, and 923,780, abandoned),the OP-3 proteins, like the OP-2 proteins, also has one additionalcysteine residue (residue 338 of Seq. ID No. 1) in the conservedC-terminal domain.

The mature sequence of OP-3 shares significant amino acid sequencehomology with the morphogens identified to date. Specifically, the sevencysteine fragment shows greater than 79% amino acid identity with thecorresponding mOP-2 and hOP-2 sequences, and greater than 66% identitywith the corresponding OP-1 sequences. Like OP-2, OP-3 has an eighthcysteine within the seven cysteine domain (e.g., at position 338 of Seq.ID No. 1). In addition, OP-3 is unique among the morphogens identifiedto date in that the residue at position 9 in the conserved sevencysteine domain (e.g., residue 315 of Seq. ID No. 1) is a serine,whereas other morphogens typically have a tryptophan at this location(see Table I below, and Table II in PCT/US92/07358, for example.)

Thus, useful OP-3 mutant variants include, but are not limited to, aminoacid sequences derived from Seq. ID No. 1 and wherein the cysteine atposition 338 is replaced with another amino acid, preferably a tyrosine,histidine, isoleucine or serine and conservative substitutions thereof,e.g., such as defined by Dayoff et al., Atlas of Protein Sequence andStructure; vol. 5, Suppl. 3, pp.345-362 (M. O. Dayoff, ed., Nat'lBioMed. Research Fdn., Washington D.C. 1979.). Still other useful OP-3mutant variants include proteins wherein the serine at position 315 isreplaced with another amino acid, preferably a tryptophan andconservative substitutions thereof.

Generic Sequence 7 (Seq. ID No. 12) and Generic Sequence 8 (Seq. ID No.13) disclosed below, accommodate the homologies shared among perferredmorphogen protein family members identified to date, including OP-1,OP-2, OP-3, CBMP2A, CBMP2B, BMP3, 60A, DPP, Vg1, BMP5, BMP6, Vrg-1, andGDF-1. The amino acid sequences for these proteins are described herein(see Sequence Listing and Table I below) and/or in the art, as well asin PCT publication US 92/07358, filed Aug. 28, 1992, for example. Thegeneric sequences include both the amino acid identity shared by thesesequences in the C-terminal domain, defined by the six and sevencysteine skeletons (Generic Sequences 7 and 8, respectively), as well asalternative residues for the variable positions within the sequence. Thegeneric sequences allow for an additional cysteine at position 41(Generic Sequence 7) or position 46 (Generic Sequence 8), providing anappropriate cysteine skeleton where inter- or intramolecular disulfidebonds can form, and containing certain critical amino acids whichinfluence the tertiary structure of the proteins. ##STR1## wherein eachXaa is independently selected from a group of one or more specifiedamino acids defined as follows: "Res." means "residue" and Xaa at res.2=(Tyr or Lys); Xaa at res. 3=Val or Ile); Xaa at res. 4=(Ser. Asp orGlu); Xaa at res. 6=(Arg, Gln, Ser, Lys or Ala); Xaa at res. 7=(Asp orGlu); Xaa at res. 8=(Leu, Val or Ile); Xaa at res. 11=(Gln, Leu, Asp,His, Asn or Ser); Xaa at res. 12=(Asp, Arg, Asn or Glu); Xaa at res.13=(Trp or Ser); Xaa at res. 14=(Ile or Val); Xaa at res. 15=(Ile orVal); Xaa at res. 16 (Ala or Ser); Xaa at res. 18=(Glu, Gln, Leu, Lys,Pro or Arg); Xaa at res. 19=(Gly or Ser); Xaa at res. 20=(Tyr or Phe);Xaa at res. 21=(Ala, Ser, Asp, Met, His, Gln, Leu or Gly); Xaa at res.23=(Tyr, Asn or Phe); Xaa at res. 26=(Glu, His, Tyr, Asp, Gln, Ala orSer); Xaa at res. 28=(Glu, Lys, Asp, Gln or Ala); Xaa at res. 30=(Ala,Ser, Pro, Gln, Ile or Asn); Xaa at res. 31=(Phe, Leu or Tyr); Xaa atres. 33=(Leu, Val or Met); Xaa at res. 34=(Asn, Asp, Ala, Thr or Pro);Xaa at res. 35=(Ser, Asp, Glu, Leu, Ala or Lys); Xaa at res. 36=(Tyr,Cys, His, Ser or Ile); Xaa at res. 37=(Met, Phe, Gly or Leu); Xaa atres. 38=(Asn, Ser or Lys); Xaa at res. 39=(Ala, Ser, Gly or Pro); Xaa atres. 40=(Thr, Leu or Ser); Xaa at res. 44=(Ile, Val or Thr); Xaa at res.45=(Val, Leu, Met or Ile); Xaa at res. 46=(Gln or Arg); Xaa at res.47=(Thr, Ala or Ser); Xaa at res. 48=(Leu or Ile); Xaa at res. 49=(Valor Met); Xaa at res. 50=(His, Asn or Arg); Xaa at res. 51=(Phe, Leu,Asn, Ser, Ala or Val); Xaa at res. 52=(Ile, Met, Asn, Ala, Val, Gly orLeu); Xaa at res. 53=(Asn, Lys, Ala, Glu, Gly or Phe); Xaa at res.54=(Pro, Ser or Val); Xaa at res. 55=(Gilu, Asp, Asn, Gly, Val, Pro orLys); Xaa at res. 56=(Thr, Ala, Val, Lys, Asp, Tyr, Ser, Gly, Ile orHis); Xaa at res. 57=(Val, Ala or Ile); Xaa at res. 58=(Pro or Asp); Xaaat res. 59=(Lys, Leu or Glu); Xaa at res. 60=(Pro, Val or Ala); Xaa atres. 63=(Ala or Val); Xaa at res. 65=(Thr, Ala or Glu); Xaa at res.66=(Gln, Lys, Arg or Glu); Xaa at res. 67=(Leu, Met or Val); Xaa at res.68=(Asn, Ser, Asp or Gly); Xaa at res. 69=(Ala, Pro or Ser); Xaa at res.70=(Ile, Thr, Val or Leu); Xaa at res. 71=(Ser, Ala or Pro); Xaa at res.72=(Val, Leu, Met or Ile); Xaa at res. 74=(Tyr or Phe); Xaa at res.75=(Phe, Tyr, Leu or His); Xaa at res. 76=(Asp, Asn or Leu); Xaa at res.77=(Asp, Glu, Asn, Arg or Ser); Xaa at res. 78=(Ser, Gln, Asn, Tyr orAsp); Xaa at res. 79=(Ser, Asn, Asp, Glu or Lys); Xaa at res. 80=(Asn,Thr or Lys); Xaa at res. 82=(Ile, Val or Asn); Xaa at res. 84=(Lys orArg); Xaa at res. 85=(Lys, Asn, Gln, His, Arg or Val); Xaa at res.86=(Tyr, Glu or His); Xaa at res. 87=(Arg, Gln, Glu or Pro); Xaa at res.88=(Asn, Glu, Trp or Asp); Xaa at res. 90=(Val, Thr, Ala or Ile); Xaa atres. 92=(Arg, Lys, Val, Asp, Gln or Glu); Xaa at res. 93=(Ala, Gly, Gluor Ser); Xaa at res. 95=(Gly or Ala) and Xaa at res. 97=(His or Arg).

As described above, Generic Sequence 8 (Seq. ID No. 13) includes all ofGeneric Sequence 7 and in addition includes the following sequence atits N-terminus:

    Cys Xaa Xaa Xaa Xaa

Accordingly, beginning with residue 7, each "Xaa" in Generic Seq. 8 is aspecified amino acid defined as for Generic Seq. 7, with the distinctionthat each residue number described for Generic Sequence 7 is shifted byfive in Generic Seq. 8. Thus, "Xaa at res. 2=(Tyr or Lys)" in Gen. Seq.7 refers to Xaa at res. 7 in Generic Seq. 8. In Generic Seq. 8, Xaa atres. 2=(Lys, Arg, Ala or Gln); Xaa at res. 3=(Lys, Arg or Met); Xaa atres. 4=(His, Arg or Gln); and Xaa at res. 5=(Glu, Ser, His, Gly, Arg,Pro, Thr, or Tyr).

Table I, set forth below, compares the C-terminal amino acid sequencesdefining the seven cysteine skeleton of human OP-1, mouse OP-1, humanOP-2, mouse OP-2, and mouse OP-3 (mOP-3, Seq. ID No. 1). In the table,the sequences are aligned essentially following the method of Needlemanet al. (1970) J. Mol. Biol., 48:443-453, calculated using the AlignProgram (DNAstar, Inc.) In the table, three dots indicates that theamino acid in that position is the same as the amino acid in hOP-1.Three dashes indicate that no amino acid is present in that position,and are included for purposes of illustrating homologies. As is apparentfrom the following amino acid sequence comparisons, significant aminoacid sequence homology exists between mouse OP-3 and mouse and humanOP-1 and OP-2.

                  TABLE I                                                         ______________________________________                                        hOP-1 Cys    Lys    Lys  His  Glu  Leu  Tyr  Val                              mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                            hOP-2 . . .  Arg    Arg  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                            mOP-2 . . .  Arg    Arg  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                            mOP-3 . . .  Arg    Arg  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                   1                       5                                              hOP-1 Ser    Phe    Arg  Asp  Leu  Gly  Trp  Gln  Asp                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       hOP-2 . . .  . . .  Gln  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Leu  . . .                       mOP-2 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Leu  . . .                       mOP-3 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Leu  . . .                                    10                         15                                    hOP-1 Trp    Ile    Ile  Ala  Pro  Glu  Gly  Tyr  Ala                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       hOP-2 . . .  Val    . . .                                                                              . . .                                                                              . . .                                                                              Gln  . . .                                                                              . . .                                                                              Ser                         mOP-2 . . .  Val    . . .                                                                              . . .                                                                              . . .                                                                              Gln  . . .                                                                              . . .                                                                              Ser                         mOP-3 Ser    Val    . . .                                                                              . . .                                                                              . . .                                                                              Gln  . . .                                                                              . . .                                                                              Ser                                             20                       25                               hOP-1 Ala    Tyr    Tyr  Cys  Glu  Gly  Glu  Cys  Ala                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       hOP-2 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Ser                         mOP-2 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       mOP-3 . . .  . . .  . . .                                                                              . . .                                                                              Ala  . . .                                                                              . . .                                                                              . . .                                                                              Ile                                                  30                       35                          hOP-1 Phe    Pro    Leu  Asn  Ser  Tyr  Met  Asn  Ala                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       hOP-2 . . .  . . .  . . .                                                                              Asp  . . .                                                                              Cys  . . .                                                                              . . .                                                                              . . .                       mOP-2 . . .  . . .  . . .                                                                              Asp  . . .                                                                              Cys  . . .                                                                              . . .                                                                              . . .                       mOP-3 Tyr    . . .  . . .                                                                              . . .                                                                              . . .                                                                              Cys  . . .                                                                              . . .                                                                              Ser                                                       40                                              hOP-1 Thr    Asn    His  Ala  Ile  Val  Gln  Thr  Leu                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       hOP-2 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              Leu  . . .                                                                              Ser  . . .                       mOP-2 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              Leu  . . .                                                                              Ser  . . .                       mOP-3 . . .  . . .  . . .                                                                              . . .                                                                              Thr  Met  . . .                                                                              Ala  . . .                             45                           50                                         hOP-1 Val    His    Phe  Ile  Asn  Pro  Glu  Thr  Val                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Asp  . . .                                                                              . . .                       hOP-2 . . .  . . .  Leu  Met  Lys  . . .                                                                              Asn  Ala  . . .                       mOP-2 . . .  . . .  Leu  Met  Lys  . . .                                                                              Asp  Val  . . .                       mOP-3 . . .  . . .  Leu  Met  Lys  . . .                                                                              Asp  Ile  Ile                                      55                         60                                    hOP-1 Pro    Lys    Pro  Cys  Cys  Ala  Pro  Thr  Gln                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       hOP-2 . . .  . . .  Ala  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Lys                         mOP-2 . . .  . . .  Ala  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Lys                         mOP-3 . . .  . . .  Val  . . .                                                                              . . .                                                                              Val  . . .                                                                              . . .                                                                              Glu                                             65                       70                               hOP-1 Leu    Asn    Ala  Ile  Ser  Val  Leu  Tyr  Phe                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       hOP-2 . . .  Ser    . . .                                                                              Thr  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Tyr                         mOP-2 . . .  Ser    . . .                                                                              Thr  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Tyr                         mOP-3 . . .  Ser    . . .                                                                              . . .                                                                              . . .                                                                              Leu  . . .                                                                              . . .                                                                              Tyr                                                  75                       80                          hOP-1 Asp    Asp    Ser  Ser  Asn  Val  Ile  Leu  Lys                         mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                       hOP-2 . . .  Ser    . . .                                                                              Asn  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Arg                         mOP-2 . . .  Ser    . . .                                                                              Asn  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Arg                         mOP-3 . . .  Arg    Asn  Asn  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Arg                                                       85                                              hOP-1 Lys    Tyr    Arg  Asn  Met  Val  Val  Arg                              mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                            hOP-2 . . .  His    . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Lys                              mOP-2 . . .  His    . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Lys                              mOP-3 Arg    Glu    . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              . . .                                                                              Gln                                    90                           95                                         hOP-1 Ala    Cys    Gly  Cys  His                                             mOP-1 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                           hOP-2 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                           mOP-2 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                           mOP-3 . . .  . . .  . . .                                                                              . . .                                                                              . . .                                                               100                                                       ______________________________________                                    

II. Formulations and Methods for Administering OP-3 Protein asTherapeutic Agents

II.A OP-3 Protein Considerations

The morphogens described herein may be provided to an individual by anysuitable means, preferably directly or systemically, e.g., parenterallyor orally. Where the morphogen is to be provided directly (e.g.,locally, as by injection, to a desired tissue site), or parenterally,such as by intravenous, subcutaneous, intramuscular, intraorbital,ophthalmic, intraventricular, intracranial, intracapsular, intraspinal,intracisternal, intraperitoneal, buccal, rectal, vaginal, intranasal orby aerosol administration, the morphogen preferably comprises part of anaqueous solution. The solution is physiologically acceptable so that inaddition to delivery of the desired morphogen to the patient, thesolution does not otherwise adversely affect the patient's electrolyteand volume balance. The aqueous medium for the morphogen thus maycomprise normal physiologic saline (0.9% NaCl, 0.15M), pH 7-7.4. Theaqueous solution containing the morphogen can be made, for example, bydissolving the protein in 50% ethanol, or acetonitrile containing 0.1%trifluoroacetic acid (TFA) or 0.1% HCl, or equivalent solvents. Onevolume of the resultant solution then is added, for example, to tenvolumes of phosphate buffered saline (PBS), which further may include0.1-0.2% human serum albumin (HSA). The resultant solution preferably isvortexed extensively.

If desired, a given morphogen may be made more soluble by associationwith a suitable molecule. For example, association of the mature dimerwith the pro domain of the morphogen increases solubility of the proteinsignificantly. For example,the pro form of OP-3 comprises a species thatis soluble in physiologically buffered solutions. In fact, theendogenous protein is thought to be transported (e.g., secreted andcirculated) to particular tissues in this form. This soluble form of theprotein may be obtained from the culture medium of morphogen-secretingmammalian cells. Alternatively, a soluble species may be formulated bycomplexing the mature dimer (or an active fragment thereof) with part orall of a pro domain. Another molecule capable of enhancing solubilityand particularly useful for oral administrations, is casein. Forexample, addition of 0.2% casein increases solubility of the matureactive form of OP-1 by 80%. Other components found in milk and/orvarious serum proteins also may be useful.

Useful solutions for oral or parenteral administration may be preparedby any of the methods well known in the pharmaceutical art, described,for example, in Remington's Pharmaceutical Sciences, (Gennaro, A., ed.),Mack Pub., 1990. Formulations may include, for example, polyalkyleneglycols such as polyethylene glycol, oils of vegetable origin,hydrogenated naphthalenes, and the like. Formulations for directadministration, in particular, may include glycerol and othercompositions of high viscosity. Biocompatible, preferably bioresorbablepolymers, including, for example, hyaluronic acid, collagen, tricalciumphosphate, polybutyrate, polylactide, polyglycolide andlactide/glycolide copolymers, may be useful excipients to control therelease of the morphogen in vivo.

Other potentially useful parenteral delivery systems for thesemorphogens include ethylene-vinyl acetate copolymer particles, osmoticpumps, implantable infusion systems, and liposomes. Formulations forinhalation administration may contain as excipients, for example,lactose, or may be aqueous solutions containing, for example,polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or oilysolutions for administration in the form of nasal drops, or as a gel tobe applied intranasally. Formulations for parenteral administration mayalso include glycocholate for buccal administration, methoxysalicylatefor rectal administration, or cutric acid for vaginal administration.

Alternatively, the morphogens described herein may be administeredorally. Oral administration of proteins as therapeutics generally is notpracticed as most proteins readily are degraded by digestive enzymes andacids in the mammalian digestive system before they can be absorbed intothe bloodstream. However, the morphogens described herein typically areacid-stable and protease-resistant (see, for example, U.S. Pat. No.4,968,590.) In addition, at least one morphogen, OP-1, has beenidentified in bovine mammary gland extract, colostrum and milk, as wellas saliva. Moreover, the OP-1 purified from mammary gland extract ismorphogenically active. For example, this protein induces endochondralbone formation in mammals when implanted subcutaneously in associationwith a suitable matrix material, using a standard in vivo bone assay,such as is disclosed in U.S. Pat. No. 4,968,590. In addition, endogenousmorphogen also is detected in human serum. These findings indicate thatoral and parenteral administration are viable means for administeringmorphogens to an individual. Moreover, while the mature forms of certainmorphogens described herein typically are sparingly soluble, themorphogen form found in milk (and mammary gland extract and colostrum)is readily soluble, probably by association of the mature,morphogenically active form with the pro domain of the intact sequenceand/or by association with one or more milk components. Accordingly, thecompounds provided herein also may be associated with molecules capableof enhancing their solubility in vitro or in vivo, including, forexample, part or all of a morphogen pro domain, and casein, as describedabove.

The compounds provided herein also may be associated with moleculescapable of targeting the morphogen to a desired tissue. For example,tetracycline and diphosphonates (bisphosphonates) are known to bind tobone mineral, particularly at zones of bone remodeling, when they areprovided systemically in a mammal. Accordingly, these molecules may beincluded as useful agents for targeting OP-3 to bone tissue.Alternatively, an antibody or other binding protein that interactsspecifically with a surface molecule on the desired target tissue cellsalso may be used. Such targeting molecules further may be covalentlyassociated to the morphogen, e.g., by chemical crosslinking, or by usingstandard genetic engineering means to create, for example, an acidlabile bond such as an Asp-Pro linkage. Useful targeting molecules maybe designed, for example, using the single chain binding site technologydisclosed, for example, in U.S. Pat. No. 5,091,513.

As described above, the morphogen family members share significantsequence homology in the C-terminal active domains. By contrast, thesequences diverge significantly in the sequences which define the prodomain and the N-terminal 39 amino acids of the mature protein.Accordingly, the pro domain and/or N-terminal sequence may bemorphogen-specific. As described above, it also is known that thevarious morphbgens identified to date are differentially expressed inthe different tissues. Accordingly, without being limited to any giventheory, it is likely that, under natural conditions in the body,selected morphogens typically act on a given tissue. Accordingly, partor all of morphogen-specific sequences may serve as targeting moleculesfor the morphogens described herein. For example, the pro domains mayinteract specifically with one or more molecules at the target tissue todirect the morphogen associated with the pro domain to that tissue.Thus, another useful targeting molecule for targeting OP-3 to bonetissue, for example, may include part or all of a morphogen-specificsequence, such as part or all of a pro domain and/or the N-terminus ofthe mature protein. Particularly useful are the morphogen-specificsequences of OP-1, BMP2 or BMP4, all of which proteins are foundnaturally associated with bone tissue (see, for example, U.S. Pat. No.5,011,691). Alternatively, the morphogen-specific sequences of GDF-1 maybe used to target morphogenic OP-3 to nerve tissue, particularly braintissue where GDF-1 appears to be primarily expressed (see, for example,U.S. Ser. No. 922,813, abandoned, and Lee, PNAS, 88:4250-4254 (1991),incorporated herein by reference). As described above, pro forms of theproteins may be obtained from the culture medium of morphogen-secretingmammalian cells. Alternatively, a suitable species may be formulated bycomplexing the mature dimer (or an active fragment thereof) with part orall of a pro domain. Chimeric OP-3 proteins comprising, for example,non-OP-3 pro domains and/or non-OP-3 N-termini, may be synthesized usingstandard recombinant DNA methodology and/or automated chemical nucleicacid synthesis methodology well described in the art and as disclosedbelow.

Finally, the OP-3 proteins provided herein may be administered alone orin combination with other molecules known to have a beneficial effect ontissue morphogenesis, including molecules capable of tissue repair andregeneration and/or inhibiting inflammation. Examples of usefulcofactors for stimulating bone tissue growth in osteoporoticindividuals, for example, include but are not limited to, vitamin D₃,calcitonin, prostaglandins, parathyroid hormone, dexamethasone, estrogenand IGF-I or IGF-II. Useful cofactors for nerve tissue repair andregeneration may include nerve growth factors. Other useful cofactorsinclude symptom-alleviating cofactors, including antiseptics,antibiotics, antiviral and antifungal agents and analgesics andanesthetics.

The compounds provided herein can be formulated into pharmaceuticalcompositions by admixture with pharmaceutically acceptable nontoxicexcipients and carriers. As noted above, such compositions may beprepared for parenteral administration, particularly in the form ofliquid solutions or suspensions; for oral administration, particularlyin the form of tablets or capsules; or intranasally, particularly in theform of powders, nasal drops or aerosols. Where adhesion to a tissuesurface is desired the composition may include the morphogen dispersedin a fibrinogen-thrombin composition or other bioadhesive such as isdisclosed, for example in PCT US91/09275, the disclosure of which isincorporated herein by reference. The composition then may be painted,sprayed or otherwise applied to the desired tissue surface.

The compositions can be formulated for parenteral or oral administrationto humans or other mammals in therapeutically effective amounts, e.g.,amounts which provide appropriate concentrations of OP-3 to targettissue for a time sufficient to induce morphogenesis, includingparticular steps thereof, as described above.

Where OP-3 is to be used as part of a transplant procedure, themorphogen may be provided to the living tissue or organ to betransplanted prior to removal of tissue or organ from the donor. OP-3may be provided to the donor host directly, as by injection of aformulation comprising OP-3 into the tissue, or indirectly, e.g., byoral or parenteral administration, using any of the means describedabove.

Alternatively or, in addition, once removed from the donor, the organ orliving tissue may be placed in a preservation solution containing OP-3.In addition, the recipient also preferably is provided with themorphogen just prior to, or concommitant with, transplantation. In allcases, OP-3 may be administered directly to the tissue at risk, as byinjection to the tissue, or it may be provided systemically, either byoral or parenteral administration, using any of the methods andformulations described herein and/or known in the art.

Where OP-3 comprises part of a tissue or organ preservation solution,any commercially available preservation solution may be used toadvantage. For example, useful solutions known in the art includeCollins solution, Wisconsin solution, Belzer solution, Eurocollinssolution and lactated Ringer's solution. Generally, an organpreservation solution usually possesses one or more of the followingproperties: (a) an osmotic pressure substantially equal to that of theinside of a mammalian cell,(solutions typically are hyperosmolar andhave K+ and/or Mg++ ions present in an amount sufficient to produce anosmotic pressure slightly higher than the inside of a mammalian cell);(b) the solution typically is capable of maintaining substantiallynormal ATP levels in the cells; and (c) the solution usually allowsoptimum maintenance of glucose metabolism in the cells. organpreservation solutions also may contain anticoagulants, energy sourcessuch as glucose, fructose and other sugars, metabolites, heavy metalchelators, glycerol and other materials of high viscosity to enhancesurvival at low temperatures, free oxygen radical inhibiting and/orscavenging agents and a pH indicator. A detailed description ofpreservation solutions and useful components may be found, for example,in U.S. Pat. No. 5,002,965, the disclosure of which is incorporatedherein by reference.

OP-3 is envisioned to be useful in enhancing viability of any organ orliving tissue to be transplanted. The morphogens may be used toparticular advantage in lung, heart, liver, kidney or pancreastransplants, as well as in the transplantation and/or grafting of bonemarrow, skin, gastrointestinal mucosa, and other living tissues.

As will be appreciated by those skilled in the art, the concentration ofthe compounds described in a therapeutic composition will vary dependingupon a number of factors, including the dosage of the drug to beadministered, the chemical characteristics (e.g., hydrophobicity) of thecompounds employed, and the route of administration. The preferreddosage of drug to be administered also is likely to depend on suchvariables as the type and extent of tissue loss or defect, the overallhealth status of the particular patient, the relative biologicalefficacy of the compound selected, the formulation of the compound, thepresence and types of excipients in the formulation, and the route ofadministration. In general terms, the compounds of this invention may beprovided in an aqueous physiological buffer solution containing about0.001 to 10% w/v compound for parenteral administration. Typical doseranges are from about 10 ng/kg to about 1 g/kg of body weight per day; apreferred dose range is from about 0.1 μg/kg to 100 mg/kg of bodyweight. No obvious morphogen-induced pathological lesions are inducedwhen mature morphogen (e.g., OP-1, 20 μg) is administered daily tonormal growing rats for 21 consecutive days. Moreover, 10 μg systemicinjections of morphogen (e.g., OP-1) injected daily for 10 days intonormal newborn mice does not produce any gross abnormalities.

II.B Matrix Preparation

A morphogenically active fragment of OP-3 may be implanted surgically,dispersed in a biocompatible, preferably in vivo biodegradable matrixappropriately modified to provide a structure or scaffold in which theOP-3 may be dispersed and which allows the differentiation andproliferation of migrating progenitor cells. The matrix also may providesignals capable of directing the tissue specificity of thedifferentiating cells, as well as providing a morphogenically permissiveenvironment, being essentially free of growth inhibiting signals.

The formulated matrix may be shaped as desired in anticipation ofsurgery or may be shaped by the physician or technician during surgery.Thus, the material may be used in topical, subcutaneous,intraperitoneal, or intramuscular implants to repair tissue or to induceits growth de novo. The matrix preferably is biodegradable in vivo,being slowly absorbed by the body and replaced by new tissue growth, inthe shape or very nearly in the shape of the implant. The matrix alsomay be particulate in nature.

Details of how to make and how to use the matrices useful in thisinvention are disclosed below and in co-pending U.S. Ser. No. 752,764,abandoned, the disclosure of which is incorporated herein by reference.

II.B(i) Tissue-Derived Matrices

Suitable biocompatible, in vivo biodegradable acellular matrices may beprepared from naturally-occurring tissue. The tissue is treated withsuitable agents to substantially extract the cellular, nonstructuralcomponents of the tissue. The agents also should be capable ofextracting any morphogenesis inhibiting components associated with thetissue. The resulting material is a porous, acellular matrix,substantially depleted in nonstructurally-associated components.

The matrix also may be further treated with agents that modify thematrix, increasing the number of pores and micropits on its surfaces.Those skilled in the art will know how to determine which agents arebest suited to the extraction of nonstructural components for differenttissues. For example, soft tissues such as liver and lung may bethin-sectioned and exposed to a nonpolar solvent such as, for example,100% ethanol, to destroy the cellular structure of the tissue andextract nonstructural components. The material then may be dried andpulverized to yield nonadherent porous particles or it may be maintainedas a gel-like solution. Structural tissues such as cartilage and dentinwhere collagen is a primary proteinaceous component may be demineralizedand extracted with guanidinium hydrochloride, essentially following themethod of Sampath et al. (1983) PNAS 80:6591-6595. For example,pulverized and demineralized dentin is extracted with five volumes of 4Mguanidinium-HCl, 50 mM Tris-HCl, pH 7.0 for 16 hours at 4° C. Thesuspension then is filtered. The insoluble material that remains iscollected and used to fabricate the matrix. The material is mostlycollagenous in matter. It is devoid of morphogenic activity. The matrixparticles may further be treated with a collagen fibril-modifying agentthat extracts potentially unwanted components from the matrix, andalters the surface structure of the matrix material. Useful agentsinclude acids, organic solvents or heated aqueous media. A detaileddescription of these matrix treatments are disclosed, for example, inU.S. Pat. No. 4,975,526 and PCT publication US90/00912, published Sep.7, 1990 (WO90/10018).

The currently most preferred agent is a heated aqueous fibril-modifyingmedium such as water, to increase the matrix particle surface area andporosity. The currently most preferred aqueous medium is an acidicaqueous medium having a pH of less than about 4.5, e.g., within therange of about pH 2-pH 4 which may help to "swell" the collagen beforeheating. 0.1% acetic acid, which has a pH of about 3, currently is mostpreferred. 0.1M acetic acid also may be used.

Various amounts of delipidated, demineralized guanidine-extracted bonecollagen are heated in the aqueous medium (Ig matrix/30 ml aqueousmedium) under constant stirring in a water jacketed glass flask, andmaintained at a given temperature for a predetermined period of time.Preferred treatment times are about one hour, although exposure times ofbetween about 0.5 to two hours appear acceptable. The temperatureemployed is held constant at a temperature within the range of about 37°C. to 65° C. The currently preferred heat treatment temperature iswithin the range of about 45° C. to 60° C.

After the heat treatment, the matrix is filtered, washed, lyophilizedand used for implant. Where an acidic aqueous medium is used, the matrixalso is preferably neutralized prior to washing and lyophilization. Acurrently preferred neutralization buffer is a 200 mM sodium phosphatebuffer, pH 7.0. To neutralize the matrix, the matrix preferably first isallowed to cool following thermal treatment, the acidic aqueous medium(e.g., 0.1% acetic acid) then is removed and replaced with theneutralization buffer and the matrix agitated for about 30 minutes. Theneutralization buffer then may be removed and the matrix washed andlyophilized.

Other useful fibril-modifying treatments include acid treatments (e.g.,trifluoroacetic acid and hydrogen fluoride) and solvent treatments suchas dichloromethane, acetonitrile, isopropanol and chloroform, as well asparticular acid/solvent combinations.

After contact with the fibril-modifying agent, the treated matrix may bewashed to remove any extracted components, following a form of theprocedure set forth below:

1. Suspend matrix preparation in TBS (Tris-buffered saline) 1 g/200 mland stir at 4° C. for 2 hrs; or in 6M urea, 50 mM Tris-HCl, 500 mM NaCl,pH 7.0 (UTBS) or water and stir at room temperature (RT) for 30 minutes(sufficient time to neutralize the pH);

2. Centrifuge and repeat wash step; and

3. Centrifuge; discard supernatant; water wash esidue; and thenlyophilize.

Alternatively, suitable matrix materials may be btained commercially.For example, an extracellular atrix extract such as Matrigel™,(Collaborative Research, Inc., Bedford) derived from mouse sarcomacells, may be used to advantage.

II.B(ii) Synthetic Matrices

In addition to the naturally-derived tissue-specific matrices describedabove, useful tissue-specific matrices may be formulated synthetically.These porous biocompatible, in vivo biodegradable synthetic matrices aredisclosed in PCT publication US91/03603, published Dec. 12, 1991(WO91/18558), the disclosure of which is hereby incorporated byreference. Briefly, the matrix comprises a porous crosslinked structuralpolymer of biocompatible, biodegradable collagen and appropriate,tissue-specific glycosaminoglycans as tissue-specific cell attachmentfactors. Collagen derived from a number of sources may be suitable foruse in these synthetic matrices, including insoluble collagen,acid-soluble collagen, collagen soluble in neutral or basic aqueoussolutions, as well as those collagens which are commercially available.

Glycosaminoglycans (GAGS) or mucopolysaccharides arehexosamine-containing polysaccharides of animal origin that have atissue specific distribution, and therefore may be used to helpdetermine the tissue specificity of the morphogen-stimulateddifferentiating cells. Reaction with the GAGs also provides collagenwith another valuable property, i.e., inability to provoke an immunereaction (foreign body reaction) from an animal host.

Chemically, GAGs are made up of residues of hexosamines glycosidicallybound and alternating in a more-or-less regular manner with eitherhexouronic acid or hexose moieties (see, e.g., Dodgson et al. inCarbohydrate Metabolism and its Disorders (Dickens et al., eds.) Vol. 1,Academic Press (1968)). Useful GAGs include hyaluronic acid, heparin,heparin sulfate, chondroitin 6-sulfate, chondroitin 4-sulfate, dermatansulfate, and keratin sulfate. Other GAGs are suitable for forming thematrix described herein, and those skilled in the art will either knowor be able to ascertain other suitable GAGs using no more than routineexperimentation. For a more detailed description of mucopolysaccharides,see Aspinall, Polysaccharides, Pergamon Press, Oxford (1970). Forexample, as disclosed in U.S. application Ser. No. 529,852, abandoned,chondroitin-6-sulfate can be used where endochondral bone formation isdesired. Heparin sulfate, on the other hand, may be used to formulatesynthetic matrices for use in lung tissue repair.

Collagen can be reacted with a GAG in aqueous acidic solutions,preferably in diluted acetic acid solutions. By adding the GAG dropwiseinto the aqueous collagen dispersion, coprecipitates of tangled collagenfibrils coated with GAG results. This tangled mass of fibers then can behomogenized to form a homogeneous dispersion of fine fibers and thenfiltered and dried.

Insolubility of the collagen-GAG products can be raised to the desireddegree by covalently cross-linking these materials, which also serves toraise the resistance to resorption of these materials. In general, anycovalent cross-linking method suitable for cross-linking collagen alsois suitable for cross-linking these composite materials, althoughcrosslinking by a dehydrothermal process is preferred.

When dry, the crosslinked particles are essentially spherical, withdiameters of about 500 μm. Scanning electron miscroscopy shows pores ofabout 20 μm on the surface and 40 μm on the interior. The interior ismade up of both fibrous and sheet-like structures, providing surfacesfor cell attachment. The voids interconnect, providing access to thecells throughout the interior of the particle. The material appears tobe roughly 99.5% void volume, making the material very efficient interms of the potential cell mass that can be grown per gram ofmicrocarrier.

Another useful synthetic matrix is one formulated from biocompatible, invivo biodegradable synthetic polymers, such as those composed ofglycolic acid, lactic acid and/or butyric acid, including copolymers andderivatives thereof. These polymers are well described in the art andare available commercially. For example, polymers composed of polyacticacid (e.g., MW 100 kDa), 80% polylactide/20% glycoside or poly3-hydroxybutyric acid (e.g., MW 30 kDa) all may be purchased fromPolySciences, Inc. The polymer compositions generally are obtained inparticulate form. In addition, one can alter the morphology of thepolymer compositions, for example to increase porosity, using any of anumber of particular solvent treatments known in the art. Where themorphogen is adsorbed to the matrix surface, the steps preferably areperformed under conditions which avoid hydrolysis of the polymers (e.g.,non-aqueous conditions such as in an ethanol-trifluoro-acetic acidsolution).

The OP-3 proteins described herein can be combined and dispersed in asuitable matrix using any of the methods described below:

1. Ethanol Precipitation

Matrix is added to the morphogen dissolved in guanidine-HCl. Samples arevortexed and incubated at a low temperature. Samples are then furthervortexed. Cold absolute ethanol is added to the mixture which is thenstirred and incubated. After centrifugation (microfuge, high speed) thesupernatant is discarded. The matrix is washed with cold concentratedethanol in water and then lyophilized.

2. Acetonitrile Trifluoroacetic Acid Lyophilization

In this procedure, a morphogenically active fragment of OP-3 in anacetonitrile trifluroacetic acid (ACN/TFA) solution is added to thecarrier material. Samples are vigorously vortexed many times and thenlyophilized.

3. Buffered Saline Lyophilization

A preparation of a morphogenically active fragment of OP-3 inphysiological saline also may be vortexed with the matrix andlyophilized to produce morphogenically active material.

Tissue morphogenesis requires a morphogenically permissive environment.Clearly, in fully-functioning healthy tissue that is not composed of apermanently renewing cell population, there must exist signals toprevent continued tissue growth. Thus, it is postulated that thereexists a control mechanism, such as a feedback control mechanism, whichregulates the control of cell growth and differentiation. In fact, it isknown that both TGF-β, and MIS are capable of inhibiting cell growthwhen present at appropriate concentrations. In addition, using the bonemodel system it can be shown that osteogenic devices comprising abone-derived carrier (matrix) that has been demineralized andguanidine-extracted to substantially remove the noncollagenous proteinsdoes allow endochondral bone formation when implanted in associationwith an osteoinductive morphogen. If, however, the bone-derived carrieris not demineralized but rather is washed only in low salt, for example,induction of endochondral bone formation is inhibited, suggesting thepresence of one or more inhibiting factors within the carrier.

III. Examples

Example 1 Recombinant Production of OP-3

OP-3 proteins useful in the methods and compositions of this inventionmay be purified from natural sources or produced using standardrecombinant methodology. General considerations for the recombinantproduction of morphogens are described in U.S. Ser. No. 752,764,abandoned, the disclosure of which is incorporated hereinabove byreference.

A. Identification of Novel mOP-3 Sequences

A genetic sequence encoding the morphogenic OP-3 protein was identifiedusing a 0.3 kb EcoRI-BamHl OP-2 fragment from a mouse OP-2 cDNA as ahybridization probe, specific to the mid-pro region of OP-2(corresponding to amino acid residues 125 to 225 of the pre-pro protein)essentially as described in U.S. Ser. No. 667,274, abandoned. The ³²P-labeled probe was prepared using the random hexanucleotide primingmethod, and the hybridizations were performed using the followingconditions: 40% formamide, 5×SSPE, 5×Denhardt's Solution, 0.1% SDS, at37° C. overnight, and washing in 0.1×SSPE, 0.1% SDS at 50° C.Approximately 1×10⁶ phages from a mouse cDNA (carried in lambda zapII)library made from the teratocarcinoma cell line PCC4 (Stratagene, Inc.,La Jolla, Calif., cat #936301) were screened. This screening yieldedfour individual clones which were purified over three rounds ofscreening. The plasmid DNA containing the cDNAs was obtained using thelambda zapII excision process following manufacturer's directions. Threeof the four clones were shown by DNA sequencing to encode OP-3. The DNAsequence, referred to herein as mOP-3 and described in Seq. ID No. 1,was identified by this procedure.

The isolated mOP-3 DNA sequence, in accordance with other knownmorphogens, encodes a protein comprising a "pro" region (definedessentially by residues 20-260 or 20-263 of Seq. ID No. 1) and a matureregion (defined essentially by residues 261-399 or 264-399 of Seq. IDNo. 1), including a functional domain comprising the conserved cysteineskeleton.

Like OP-2, OP-3 is marked by an eighth cysteine within the sevencysteine domain (e.g., at position 338 of Seq. ID No.1). The extracysteine likely helps stabilize the folded structure, possibly byproviding inter-molecular disulfide bonding. The extra cysteine alsoallows for heterodimer formation between OP-3 and another morphogencomprising the "eighth" cysteine, like OP-2 for example, or a modifiedOP-1, wherein an extra cysteine has been inserted at the appropriatelocation. The extra cysteine also may allow tetramer formation. Theextra cysteine does not inhibit synthesis or reduce the stability of thetranslated sequence significantly as expressed proteins comprising theextra cysteine are readily detected by SDS gel electrophoresis. Aprimary glycosylation site occurs just C terminal to the extra cysteinein both OP-2 and OP-3, which may provide a protective effect.

The CDNA sequences for both human and mouse OP-2 are provided in Seq. IDNos. 7 and 9, and the genomic sequence for human OP-2 is provided inSeq. ID No. 11 SEQ ID NO: 14 and SEQ ID: 15, wherein the exons definingthe coding region of these proteins are indicated. The exon boundariesalso are indicated in FIG. 1, described below. The human OP-2 locus wasisolated from a genomic library (Clontech, EMBL-3 #HL1067J) on threeoverlapping phage clones, using standard cloning procedures. The OP-2coding information was spread over 27 kb and, like OP-1, contains 7exons. A comparison of exon-intron boundaries in the 7 cysteine domainshowed matching locations with those of OP-1. The first OP-2 exoncontains 334 bp of coding sequence (111 amino acids), including thesignal peptide, and is followed by the largest intron (14.6 kb). Thesecond exon (190 bp, 64 amino acids) is separated by a short intron (0.4kb) from exon 3 (149 bp, 49 amino acids). It follows a large thirdintron of 9.5 kb. The fourth exon (195 bp, 65 amino acids) encodes thematuration site ("OP-2-Ala") and is followed by a 0.8 kb intron. The 7cysteine domains resides on exons 5 to 7: exon 5 (80 bp, 27 amino acids)encodes the first cysteine of mature OP-2 and is followed by intron 5(0.5 kb in length), exon 6 (111 bp, 37 amino acids) is separated by a2.5 kb intron from the seventh, last exon with 147 bp (49 amino acids)of coding sequence. As stated above, the exon-intron boundaries areconserved between human OP-1 and OP-2, two different members of themorphogen family of proteins. By analogy, the exon-intron boundariesbetween human and mouse OP-2, two species variants of a morphogen, areanticipated to be conserved as well.

FIG. 1 shows the alignment of the murine OP-2 (bases 93-1289 of SEQ IDNO:9) and murine OP-3 (bases 69-1265 of SEQ ID NO:1) coding regions ofthe cDNA. The exon boundaries are indicated by bars beneath thesequence. Both sequences have the same number of nucleotides. Thenucleotide sequence is about 80% conserved in the N-terminal andC-terminal regions. In the figure, nucleotide identity between thesequences is indicated by stippling. In addition, the central region ofthe sequence is highly conserved and this conserved region falls intothe boundaries of exon 2 and 3. There are only threenucleotideidifferences in this region, indicated in the figure bydiamonds.

The high degree of conservation in the nucleotide sequences indicatesthat OP-2 and OP-3 likely share the nucleotide sequence of exon 2 and 3.The different proteins may result from alternatively splicedtranscripts, or they may arise from independent genes which share partof their coding sequence. Intron 1, which lies upstream of exon 2 inOP-2 (see Seq. ID No. 11 and SEQ ID NO. 14) is large (14.6 kb) and couldinclude the start of the OP-3 gene and/or its first exon sequence.Certainly, as has been found for other mammalian genes, one or more ofthe introns of these morphogens may include sequences having atranscription regulatory function.

Using the screening procedure described herein and in U.S. Ser. No.752,764, abandoned, and the labelled OP-2 fragment, or preferably alabelled OP-3 fragment, OP-3 genetic sequences from other species andother libraries may be isolated. Alternatively, or in addition, a probeto the N-terminal region of the mature protein, or the 3' noncodingregion flanking and immediately following the stop codon, also may beused to screen for other OP-3 species variants. These sequences varysubstantially among the morphogens and represent morphogen-specificsequences. Mammalian cell expression of OP-3 readily can be achievedusing COS (simian kidney ATCC, CRL-1650) or

CHO (Chinese hamster ovary) cells (e.g., CHO-DXBII, from LawrenceChasin, Columbia University, NY). An exemplary protocol for mammaliancell expression is provided below. Other useful eukaryotic cell systemsinclude the insect/baculovirus system or the mammalian complementsystem.

B. Expression of Novel OP-3 Sequences

To express the OP-3 protein, the OP-3 DNA is subcloned into an insertionsite of a suitable, commercially available pUC-type vector (e.g.,pUC-19, ATCC #37254, Mannassas, Va.), along with a suitablepromoter/enhancer sequences and 3' termination sequences. Currentlypreferred promoter/enhancer sequences are the CMV promoter (humancytomegalovirus major intermediate--early promoter) and the mousemammary tumor virus promoter (mMTV) boosted by the rous sarcoma virusLTR enhancer sequence (e.g., from Clontech, Inc., Palo Alto). Expressionalso may be further enhanced using transactivating enhancer sequences.The plasmid also contains DHFR as an amplifiable marker, under SV40early promoter control (ATCC #37148). Transfection, cell culturing, geneamplification and protein expression conditions are standard conditions,well known in the art, such as are described, for example in Ausubel etal., ed., Current Protocols in Molecular Biology, John Wiley & Sons, NY(1989). Briefly, transfected cells are cultured in medium containing0.1-0.5% dialyzed fetal calf serum (FCS), stably transfected highexpression cell lines obtained by subcloning and evaluated by standardNorthern blot. Southern blots also are used to assess the state ofintegrated OP-3 sequences and the extent of their copy numberamplification.

Chimeric OP-3 morphogens, e.g., comprising an OP-3 active domain and,for example, part or all of a pro domain from another, differentmorphogen may be constructed using standard recombinant DNA technologyand/or an automated DNA synthesizer to construct the desired sequence.Useful chimeras include those wherein the non-OP-3 sequence is joined tothe OP-3 sequence encoding the mature OP-3 protein, and the non-OP-3sequence encodes part or all of the sequence between the signal peptideprocessing site and the "Arg-Xaa-Xaa-Arg" processing sequence from atleast one morphogen. Alternatively, the non-OP-3 sequence may be joinedto an OP-3 sequence encoding, for example, the 6 or 7 cysteineskeletons, wherein the non-OP-3 sequence includes the sequence encodingthe N-terminus of the mature protein. As will be appreciated by personsskilled in the art, the non-OP-3 sequences may be composed of sequencesfrom one or morphogens and/or may comprise novel biosynthetic sequences.

The expressed protein then is purified as follows. For a typical 2 Lpreparation of transfected mammalian cells conditioned in 0.5% FCS, forexample, the total protein is typically about 700 mg. The amount of OP-3in the media, estimated by Western blot, is between about 0.1-5.0 mg.OP-3 media then is diluted in a low salt, physiologically buffered 6Murea solution, and loaded onto an S-Sepharose column, which acts as astrong cation exchanger. OP-3 binds to the column in low salt, and serumproteins are removed. The column subsequently is developed with an NaClgradient, e.g., 0.1M NaCl-1.0M NaCl, in 6M urea, 20 mM HEPES, pH 7.0.Most contaminants are removed at the start of the gradient, and OP-3 iseluted primarily at a higher salt concentration.

The sample then is loaded onto a phenyl-Sepharose column (hydrophobicinteraction chromatography). OP-3 binds phenyl-Sepharose in the presenceof high concentrations of a weak chaotropic salt (e.g., 1M (NH₄)₂ SO₄ ina physiologically buffered 6M urea solution). Once OP-3 is bound, thecolumn is developed with a decreasing ammonium sulfate gradient, e.g.,0.6M-0.0M (NH₄)₂ SO₄ gradient in a physiologically buffered, 6M ureasolution. Again, most contaminants are removed at the start of thegradient, and OP-3 elutes primarily at low or no ammonium sulfateconcentrations.

The OP-3 eluted from the phenyl-Sepharose column then is dialyzedagainst water, and prepared for loading onto a reverse phasechromatography column (e.g., C-18 HPLC), for example, by dialyzingagainst 30% acetonitrile, 0.1% TFA.

An alternative chromatography protocol is to perform the S-Sepharosechromatography in the absence of 6M urea. The bound proteins then areeluted with salt step elutions (e.g., 0.1-0.6M NaCl). Remaining OP-3then can be eluted in the presence of 6M urea. The 6M urea elution alsomay be used in place of the non-urea elution to achieve maximum recoveryin one step. In addition, OP-3 may be eluted from the phenyl-Sepharosecolumn in 38% ethanol-0.01% TFA, thereby eliminating the need to dialyzethe eluent before applying it to the C-18 column. Finally, multiple C-18columns may be used (e.g., three), to further enhance purification andconcentration of the protein.

OP-3 also will bind hydroxyapatite efficiently, typically in the absenceof 6 M urea and at low phosphate concentrations (less than 5 mMphosphate). Bound OP-3 can be removed from the column with an elutiongradient of about 0.001-0.5M step elution of phosphate in aphysiologically buffered solution. Additionally, urea (6M) may be addedduring the elution step.

Other related chromatography methods also may be useful in purifyingOP-3 from eucaryotic cell culture systems. For example,heparin-Sepharose may be used in combination with the S-Sepharosecolumn. Alternatively, immobilized metal-ion affinity chromatography(IMAC) (e.g., Cu²⁺ or Zn⁺) and a physiologically buffered phosphatesolution may be used to advantage.

Example 2 Mitogenic Effect of OP-3

2.1 Mitogenic Effect of Morphogen on Rat and Human Osteoblasts

The following example demonstrates the ability of OP-3 to induceproliferation of osteoblasts in vitro using the following assay. In thisand all examples involving osteoblast cultures, rat osteoblast-enrichedprimary cultures preferably are used. Although these cultures areheterogeneous in that the individual cells are at different stages ofdifferentiation, the culture is believed to more accurately reflect themetabolism and function of osteoblasts in vivo than osteoblast culturesobtained from established cell lines. Unless otherwise indicated, allchemicals referenced are standard, commercially available reagents,readily available from a number of sources, including Sigma Chemical,Co., St. Louis; Calbiochem, Corp., San Diego and Aldrich Chemical Co.,Milwaukee.

Rat osteoblast-enriched primary cultures are prepared by sequentialcollagenase digestion of newborn suture-free rat calvaria (e.g., from1-2 day-old animals, Long-Evans strain, Charles River Laboratories,Wilmington, Mass.), following standard procedures, such as aredescribed, for example, in Wong et al., (1975) PNAS 72:3167-3171. Ratosteoblast single cell suspensions then are plated onto a multi-wellplate (e.g., a 24 well plate) at a concentration of 50,000 osteoblastsper well in alpha MEM (modified Eagle's medium, Gibco, Inc., LongIsland) containing 10% FBS (fetal bovine serum), L-glutamine andpenicillin/streptomycin. The cells are incubated for 24 hours at 37° C.,at which time the growth medium is replaced with alpha MEM containing 1%FBS and the cells incubated for an additional 24 hours so that cells arein serum-deprived growth medium at the time of the experiment.

The cultured cells are divided into three groups: (1) wells whichreceive, for example, 0.1, 1.0, 10.0, 40 and 80.0 ng of OP-3; (2) wellswhich receive 0.1, 1.0, 10.0 and 40 ng of a local-acting growth factor(e.g., TGF-β); and (3) the control group, which receive no growthfactors. The cells then are incubated for an additional 18 hours afterwhich the wells are pulsed with 2 μCi/well of ³ H-thymidine andincubated for six more hours. The excess label then is washed off with acold solution of 0.15M NaCl and then 250 μl of 10% tricholoracetic acidis added to each well and the wells incubated at room temperature for 30minutes. The cells then are washed three times with cold distilledwater, and lysed by the addition of 250 μl of 1% sodium dodecyl sulfate(SDS) for a period of 30 minutes at 37° C. The resulting cell lysatesare harvested using standard means well known in the art, and theincorporation of ³ H-thymidine into cellular DNA determined by liquidscintillation as an indication of mitogenic activity of the cells. Inthe experiment, OP-3 stimulates ³ H-thymidine incorporation into DNA,and thus promote osteoblast cell proliferation. By contrast, the effectof TGF-β is transient and biphasic. At high concentrations, TGF-β has nosignificant effect on osteoblast cell proliferation.

The in vitro effect of OP-3 on osteoblast proliferation also may beevaluated using human primary osteoblasts (obtained from bone tissue ofa normal adult patient and prepared as described above) and on humanosteosarcoma-derived cell lines. In all cases OP-3 induces cellproliferation in accordance with the morphogen's ability to induceendochondral bone formation (see Example 7, below).

2.2 Progenitor Cell Stimulation

The following example demonstrates the ability ability of OP-3 tostimulate the proliferation of mesenchymal progenitor cells. Usefulnaive stem cells include pluripotential stem cells, which may beisolated from bone marrow or umbilical cord blood using conventionalmethodologies, (see, for example, Faradji et al., (1988) Vox Sang., 55(3):133-138 or Broxmeyer et al., (1989) PNAS 86:3828-3832), as well asnaive stem cells obtained from blood. Alternatively, embryonic cells(e.g., from a cultured mesodermal cell line) may be useful.

Another method for obtaining progenitor cells and for determining theability of OP-3 fragments to stimulate cell proliferation is to captureprogenitor cells from an in vivo source. For example, a biocompatiblematrix material able to allow the influx of migratory progenitor cellsmay be implanted at an in vivo site long enough to allow the influx ofmigratory progenitor cells. For example, a bone-derived,guanidine-extracted matrix, formulated as disclosed for example inSampath et al. ((1983) PNAS 80:6591-6595), or U.S. Pat. No. 4,975,526,may be implanted into a rat at a subcutaneous site, essentiallyfollowing the method of Sampath et al. After three days the implant isremoved, and the progenitor cells associated with the matrix dispersedand cultured.

Progenitor cells, however obtained, then are incubated in vitro withOP-3 under standard cell culture conditions well described in the artand described hereinabove. In the absence of external stimuli, theprogenitor cells do not, or only minimally, proliferate on their own inculture. However, progenitor cells cultured in the presence of amorphogenically active fragment of OP-3 do proliferate. Cell growth canbe determined visually or spectrophotometrically using standard methodswell known in the art.

Example 3 Morphogen-Induced Cell Differentiation

3.1 Embryonic Mesenchyme Differentiation

Morphogenically active fragments of OP-3 can be utilized to induce celldifferentiation. The ability of OP-3 to induce cell differentiation canbe demonstrated by culturing early mesenchymal cells in the presence ofOP-3 and then studying the histology of the cultured cells by stainingwith toluidine blue using standard cell culturing and cell stainingmethodologies well described in the art. For example, it is known thatrat mesenchymal cells destined to become mandibular bone, when separatedfrom the overlying epithelial cells at stage 11 and cultured in vitrounder standard tissue culture conditions, e.g., in a chemically defined,serum-free medium, containing for example, 67% DMEM (Dulbecco's modifiedEagle's medium), 22% F-12 medium, 10 mM Hepes pH 7, 2 mM glutamine, 50μg/ml transferrin, 25 μg/ml insulin, trace elements, 2 mg/ml bovineserum albumin coupled to oleic acid, with HAT (0.1 mM hypoxanthine, 10μM aminopterin, 12 μM thymidine, will not continue to differentiate.However, if these same cells are left in contact with the overlyingendoderm for an additional day, at which time they become stage 12cells, they will continue to differentiate on their own in vitro to formchondrocytes. Further differentiation into osteoblasts and, ultimately,mandibular bone, requires an appropriate local environment, e.g., avascularized environment.

Stage 11 mesenchymal cells, cultured in vitro in the presence of OP-3,e.g., 10-100 ng/ml, will continue to differentiate in vitro to formchondrocytes just as they continue to differentiate in vitro if they arecultured with the cell products harvested from the overlying endodermalcells. This experiment may be performed with different mesenchymal cellsto demonstrate the cell differentiation capability of OP-3 in differenttissues.

As another example of morphogen-induced cell differentiation, theability of OP-3 to induce osteoblast differentiation may be demonstratedin vitro using primary osteoblast cultures, or osteoblast-like cellslines, and assaying for a variety of bone cell markers that are specificmarkers for the differentiated osteoblast phenotype, e.g., alkalinephosphatase activity, parathyroid hormone-mediated cyclic AMP (cAMP)production, osteocalcin synthesis, and enhanced mineralization rates.

3.2 Alkaline Phosphatase Induction of Osteoblasts by OP-3

The cultured cells in serum-free medium are incubated with, a range ofOP-3 concentrations, for example, 0.1, 1.0, 10.0, 40.0 or 80.0 ngOP-3/ml medium; or with a similar range of TGF-β concentrations. 72hours after the incubation period the cell layer is extracted with 0.5ml of 1% Triton X-100. The resultant cell extract then, is centrifuged,and 100 pl of the extract is added to 90 μl of paranitrosophenylphospate(PNPP)/glycerine mixture and incubated for 30 minutes in a 37° C. waterbath and the reaction stopped with 100 μl NaOH. The samples then are runthrough a plate reader (e.g., Dynatech MR700 plate reader, andabsorbance measured at 400 nm, using p-nitrophenol as a standard) todetermine the presence and amount of alkaline phosphate activity.Protein concentrations are determined by the Biorad method. Alkalinephosphatase activity is calculated in units/μg protein, where 1 unit=1nmol p-nitrophenol liberated/30 minutes at 37° C.

OP-3 alone stimulates the production of alkaline phosphatase inosteoblasts, and thus promotes the growth and expression of theosteoblast differentiated phenotype.

The long term effect of OP-3 morphogen on the production of alkalinephosphatase by rat osteoblasts also may be demonstrated as follows.

Rat osteoblasts are prepared and cultured in multi-well plates asdescribed above. In this example six sets of 24 well plates are platedwith 50,000 rat osteoblasts per well. The wells in each plate, preparedas described above, then are divided into three groups: (1) those whichreceive, for example, 1 ng of OP-3 per ml of medium; (2) those whichreceive 40 ng of OP-3 per ml of medium; and (3) those which received 80ng of OP-3 per ml of medium. Each plate then is incubated for differentlengths of time: 0 hours (control time), 24 hours, 48 hours, 96 hours,120 hours and 144 hours. After each incubation period, the cell layer isextracted with 0.5 ml of 1% Triton X-100. The resultant cell extract iscentrifuged, and alkaline phosphatase activity determined as for Example3.1, using paranitroso-phenylphosphate (PNPP). OP-3 stimulates theproduction of alkaline phosphatase in osteoblasts in dose-dependentmanner so that increasing doses of OP-3 further increase the level ofalkaline phosphatase production, and moreover, the OP-3-stimulatedelevated levels of alkaline phosphatase in the treated osteoblasts isanticipated to last for an extended period of time.

3.3 OP-3 Protein Induction of PTH-Mediated cAMP.

The effect of a OP-3 on parathyroid hormone-mediated cAMP production inrat osteoblasts in vitro may be demonstrated as follows.

Rat osteoblasts are prepared and cultured in a multiwell plate asdescribed above. The cultured cells then are divided into three groups:(1) wells which receive, for example, 1.0, 10.0 and 40.0 ng OP-3/mlmedium); (2) wells which receive for example, TGF-β, at similarconcentration ranges; and (3) a control group which receives no growthfactors. The plate is then incubated for another 72 hours. At the end ofthe 72 hours the cells are treated with medium containing 0.5% bovineserum albumin (BSA) and 1 mM 3-isobutyl-1-methylxanthine for 20 minutesfollowed by the addition into half of the wells of human recombinantparathyroid hormone (hPTH, Sigma, St. Louis) at a concentration of 200ng/ml for 10 minutes. The cell layer then is extracted from each wellwith 0.5 ml of 1% Triton X-100. The cAMP levels then are determinedusing a radioimmunoassay kit (e.g., Amersham, Arlington Heights, Ill.).OP-3 alone stimulates an increase in the PTH-mediated cAMP response, andthus promotes the growth and expression of the osteoblast differentiatedphenotype.

3.4 OP-3 Protein Induction of Osteocalcin Production

Osteocalcin is a bone-specific protein synthesized by osteoblasts whichplays an integral role in the rate of bone mineralization in vivo.Circulating levels of osteocalcin in serum are used as a marker forosteoblast activity and bone formation in vivo. Induction of osteocalcinsynthesis in osteoblast-enriched cultures can be used to demonstrateOP-3 morphogenic efficacy in vitro.

Rat osteoblasts are prepared and cultured in a multi-well plate asabove. In this experiment the medium is supplemented with 10% FBS, andon day 2, cells are fed with fresh medium supplemented with fresh 10 mMβ-glycerophosphate (Sigma, Inc.). Beginning on day 5 and twice weeklythereafter, cells are fed with a complete mineralization mediumcontaining all of the above components plus fresh L(+)-ascorbate, at afinal concentration of 50 μg/ml medium. OP-3 then is added to the wellsdirectly, e.g., in 50% acetonitrile (or 50% ethanol) containing 0.1%trifluoroacetic acid (TFA), at no more than 5 μl morphogen/ml medium.Control wells receive solvent vehicle only. The cells then are re-fedand the conditioned medium sample diluted 1:1 in standardradioimmunoassay buffer containing standard protease inhibitors andstored at -20° C. until assayed for osteocalcin. Osteocalcin synthesisis measured by standard radioimmunoassay using a commercially availableosteocalcin-specific antibody.

Mineralization is determined on long term cultures (13 day) using amodified von Kossa staining technique on fixed cell layers: cells arefixed in fresh 4% paraformaldehyde at 23° C. for 10 min, followingrinsing cold 0.9% NaCl. Fixed cells then are stained for endogenousalkaline phosphatase at pH 9.5 for 10 min, using a commerciallyavailable kit (Sigma, Inc.) Purple stained cells then are dehydratedwith methanol and air dried. after 30 min incubation in 3% AgNO₃ in thedark, H₂ O-rinsed samples are exposed for 30 sec to 254 nm UV light todevelop the black silver-stained phosphate nodules. Individualmineralized foci (at least 20 μm in size) are counted under a dissectingmicroscope and expressed as nodules/culture.

OP-3 stimulates osteocalcin synthesis in osteoblast cultures. Theincreased osteocalcin synthesis in response to OP-3 is dose dependentand shows a significant increase over the basal level after 13 days ofincubation. The enhanced osteocalcin synthesis also can be confirmed bydetecting the elevated osteocalcin MRNA message (20-fold increase) usinga rat osteocalcin-specific probe. In addition, the increase inosteoclacin synthesis correlates with increased mineralization in longterm osteoblast cultures as determined by the appearance of mineralnodules. OP-3 increases the initial mineralization rate significantlycompared to untreated cultures.

3.5 Morphogen-Induced CAM Expression

The morphogens described herein induce CAM expression, particularlyN-CAM expression, as part of their induction of morphogenesis (seecopending U.S. Ser. No. 922,813, abandoned). CAMs are morphoregulatorymolecules identified in all tissues as an essential step in tissuedevelopment. N-CAMs, which comprise at least 3 isoforms (N-CAM-180,N-CAM-140 and N-CAM-120, where "180", "140" and "120" indicate theapparent molecular weights of the isoforms as measured by SDSpolyacrylamide gel electrophoresis) are expressed at least transientlyin developing tissues, and permanently in nerve tissue. Both theN-CAM-180 and N-CAM-140 isoforms are expressed in both developing andadult tissue. The N-CAM-120 isoform is found bnly in adult tissue.Another neural CAM is L1.

The ability of OP-3 to stimulate CAM expression can be demonstratedusing the following protocol, using NG108-15 cells. NG108-15 is atransformed hybrid cell line (neuroblastoma×glioma, America Type TissueCulture (ATCC), Manassas, Va.), exhibiting a morphology characteristicof transformed embryonic neurons. As described in Example 4, below,untreated NG108-15 cells exhibit a fibroblastic, or minimallydifferentiated, morphology and express only the 180 and 140 isoforms ofN-CAM normally associated with a developing cell. Following morphogentreatment these cells exhibit a morphology characteristic of adultneurons and express enhanced levels of all three N-CAM isoforms.

In this example, NG108-15 cells are cultured for 4 days in the presenceof increasing concentrations of OP-3 using standard culturingprocedures, and standard Western blots performed on whole cell extracts.N-CAM isoforms are detected with an antibody which crossreacts with allthree isoforms, mAb H28.123, obtained from Sigma Chemical Co., St.Louis, the different isoforms being distinguishable by their differentmobilities on an electrophoresis gel. Control NG108-15 cells (untreated)express both the 140 kDa and the 180 kDa isoforms, but not the 120 kDa,as determined by Western blot analyses using up to 100 μg of protein.Treatment of NG108-15 cells with OP-3 results in a dose-dependentincrease in the expression of the 180 kDa and 140 kDa isoforms, as wellas the induction of the 120 kDa isoform induced. In addition,OP-3-induced CAM expression correlates with cell aggregation, asdetermined by histology.

Example 4 OP-3 Protein-Induced Redifferentiation of TransformedPhenotype

The OP-3 morphogens described herein also can induce redifferentiationof transformed cells to a morphology characteristic of untransformedcells. The examples provided below detail morphogen-inducedredifferentiation of a transformed human cell line of neuronal origin(NG108-15); as well as mouse euroblastoma cells (N1E-115), and humanembryo arcinoma cells, to a morphology characteristic of ntransformedcells.

As described above, NG108-15 is a transformed hybrid cell line producedby fusing neuroblastoma×glioma cells (obtained from ATTC, Manassas,Va.), and exhibiting a morphology characteristic of transformedembryonic neurons, e.g., having a fibroblastic morphology. Specifically,the cells have polygonal cell bodies, short, spike-like processes andmake few contacts with neighboring cells (see copending U.S. Ser. No.922,813, abandoned). Incubation of NG108-15 cells, cultured in achemically defined, serum-free medium, with 0.1 to 300 ng/ml ofmorphogen (e.g.; OP-3) for four hours induces an orderly, dose-dependentchange in cell morphology.

In the example, NG108-15 cells are subcultured on poly-L-lysine coated 6well plates. Each well contains 40-50,000 cells in 2.5 ml of chemicallydefined medium. On the third day, 2.5 μl of morphogen (e.g., OP-3) in60% ethanol containing 0.025% trifluoroacetic is added to each well.Morphogenic OP-3 of varying concentrations are tested (typically,concentration ranges of 0-300 ng/ml are tested). The media is changeddaily with new aliquots of morphogen. OP-3 induces a dose-dependentredifferentiation of the transformed cells, including a rounding of thesoma, an increase in phase brightness, extension of the short neuriteprocesses, and other significant changes in the cellular ultrastructure.After several days treated cells begin to form epithelioid sheets thatthen become highly packed, multi-layered aggregates, as determinedvisually by microscopic examination.

Moreover, morphogen-induced redifferentiation occurs without anyassociated changes in DNA synthesis, cell division, or cell viability,making it unlikely that the morphologic changes are secondary to celldifferentiation or a toxic effect of the morphogen. In addition, themorphogen-induced redifferentiation does not inhibit cell division, asdetermined by ³ H-thymidine uptake, unlike other molecules which havebeen shown to stimulate differentiation of transformed cells, such asbutyrate, DMSO, retanoic acid or Forskolin in analogous experiments.Thus, OP-3 maintains cell stability and viability after inducingredifferentiation.

The OP-3 morphogens described herein accordingly provide usefultherapeutic agents for the treatment of neoplasias and neoplasticlesions of the nervous system, particularly in the treatment ofneuroblastomas, including retinoblastomas, and gliomas.

As yet another, related example, the ability of OP-3 to induce the"redifferentiation" of transformed human cells may be demonstrated usingthe following assay. Specifically, the effect of OP-3 on human EC cells(embryo carcinoma cells, e.g., NTERA-Z CL.D1, ATCC, Manassas, Va.) maybe determined. In the absence of an external stimulant, these cells canbe maintained as undifferentiated stem cells, and can be induced to growin serum free media (SFM). In the absence of treatment with a morphogen,the cells proliferate rampantly and are anchorage-independent. In thepresence of morphogen, EC cells grow as flattened cells, becominganchorage dependent and forming aggregates. In addition, growth rate isreduced approximately 10 fold. Ultimately, the cells are induced todifferentiate. In the example, varying concentrations of OP-3 (e.g.,0-300 ng/ml) are added daily to cultured cells (e.g., 40-50,000 cells in2.5 ml chemically defined medium), and the effects of treatmentdetermined by visual examination. OP-3 stimulates redifferentiation ofthese cells to a morphology characteristic of untransformed embryocells.

Example 5 Maintenance of Phenotype

Morphogenically active fragments of OP-3 also may be used to maintain acell's differentiated phenotype. This application is particularly usefulfor inducing the continued expression of phenotype in senescent orquiescent cells.

5.1 In Vitro Model for Phenotypic Maintenance

The phenotypic maintenance capability of morphogens is determinedreadily. A number of differentiated cells become senescent or quiescentafter multiple passages in vitro under standard tissue cultureconditions well described in the art (e.g., Culture of Animal Cells: AManual of Basic Techniques, C. R. Freshney, ed., Wiley, 1987). However,if these cells are cultivated in vitro in association with a morphogensuch as OP-3, cells are stimulated to maintain expression of theirphenotype through multiple passages. For example, the alkalinephosphatase activity of cultured osteoblasts, such as culturedosteosarcoma cells and calvaria cells, is significantly reduced aftermultiple passages in vitro. However, if the cells are cultivated in thepresence of OP-3, alkaline phosphatase activity is maintained overextended periods of time. Similarly, phenotypic expression of myocytesalso is maintained in the presence of a morphogen. In the experiment,osteoblasts are cultured as described in Example 2. The cells aredivided into groups, incubated with varying concentrations of OP-3(e.g., 0-300 ng/ml) and passaged multiple times (e.g., 3-5 times) usingstandard methodology. Passaged cells then are tested for alkalinephosphatase activity, as described in Example 3 as an indication ofdifferentiated cell metabolic function. Osteoblasts cultured in theabsence of OP-3 have reduced alkaline phosphatase activity, as comparedto OP-3-treated cells.

5.2 In Vivo Model for Phenotypic Maintenance

Phenotypic maintenance capability also may be demonstrated in vivo,using a rat model for osteoporosis, as disclosed in the copending U.S.Ser. No. 752,857, filed Aug. 30, 1991, and U.S. Ser. No. 923,780incorporated hereinabove. As described therein, Long Evans female rats(Charles River Laboratories, Wilmington, Mass.) are Sham-operated(control animals) or ovariectomized using standard surgical techniques,to produce an osteoporotic condition resulting from decreased estrogenproduction. Shortly following surgery, e.g., 200 days after ovariectomy,rats are systemically provided with phosphate buffered saline (PBS) ormorphogen, (e.g., OP-3, 1-100 μg) for 21 days (e.g., by daily tail veininjection.) The rats then are sacrificed and serum alkaline phosphataselevels, serum calcium levels, and serum osteocalcin levels aredetermined, using standard methodologies as described therein and above.Elevated levels of osteocalcin and alkaline phosphatase are observed inthe rats treated with an effective amount of OP-3. Moreover,histomorphometric analysis on the tibial diasypheal bone shows improvedbone mass in OP-3-treated animals as compared with untreated,ovariectomized rats. In fact, the bone mass of OP-3-animals iscomparable to (e.g., approaches) that of the sham-operated (e.g.,nonovarectomized) rats.

Example 6 Proliferation of Progenitor Cell Populations

Progenitor cells may be stimulated to proliferate in vivo or ex vivo.The cells may be stimulated in vivo by injecting or otherwise providinga sterile preparation containing the morphogenically active fragment ofOP-3 into the individual. For example, the hemopoietic pluripotentialstem cell population of an individual may be stimulated to proliferateby injecting or otherwise providing an appropriate concentration of OP-3to the individual's bone marrow.

Progenitor cells may be stimulated ex vivo by contacting progenitorcells of the population to be enhanced with a morphogenically activefragment of OP-3 under sterile conditions at a concentration and for atime sufficient to stimulate proliferation of the cells. Suitableconcentrations and stimulation times may be determined empirically,essentially following the procedure described in Example 2, above. Amorphogen concentration of between about 0.1-100 ng/ml and a stimulationperiod of from about 10 minutes to about 72 hours, or, more generally,about 24 hours, typically should be sufficient to stimulate a cellpopulation of about 10⁴ to 10⁶ cells. The stimulated cells then areprovided to the individual as, for example, by injecting the cells to anappropriate in vivo locus. Suitable biocompatible progenitor cells maybe obtained by any of the methods known in the art or describedhereinabove.

Example 7 Regeneration of Damaged or Diseased Tissue

OP-3 may be used to repair diseased or damaged mammalian tissue. Thetissue to be repaired preferably is assessed first, and excess necroticor interfering scar tissue removed as needed, e.g., by ablation or bysurgical, chemical, or other methods known in the medical arts.

OP-3 then may be provided directly to the tissue locus as part of asterile, biocompatible composition, either by surgical implantation orinjection. The morphogen also may be provided systemically, as by oralor parenteral administration. Alternatively, a sterile, biocompatiblecomposition containing progenitor cells stimulated by a morphogenicallyactive fragment of OP-3 may be provided to the tissue locus. Theexisting tissue at the locus, whether diseased or damaged, provides theappropriate matrix to allow the proliferation and tissue-specificdifferentiation of progenitor cells. In addition, a damaged or diseasedtissue locus, particularly one that has been further assaulted bysurgical means, provides a morphogenically permissive environment.Systemic provision of OP-3 will be sufficient for certain applications(e.g., in the treatment of osteoporosis and other disorders of the boneremodeling cycle, as an example).

In some circumstances, particularly where tissue damage is extensive,the tissue may not be capable of providing a sufficient matrix for cellinflux and proliferation. In these instances, it may be necessary toprovide OP-3 or progenitor cells stimulated by OP-3 to the tissue locusin association with a suitable, biocompatible, formulated matrix,prepared by any of the means described below. The matrix preferably isin vivo biodegradable. The matrix also may be tissue-specific and/or maycomprise porous particles having dimensions within the range of 70-850μm, most preferably 150-420 μm.

OP-3 also may be used to prevent or substantially inhibitimmune/inflammatory response-mediated tissue damage and scar tissueformation following an injury. OP-3 is provided to a newly injuredtissue locus, to induce tissue morphogenesis at the locus, preventingthe aggregation of migrating fibroblasts into non-differentiatedconnective tissue. OP-3 preferably is provided as a sterilepharmaceutical preparation injected into the tissue locus within fivehours of the injury. Where an immune/inflammatory response isunavoidably or deliberately induced, as part of, for example, a surgicalor other aggressive clinical therapy, OP-3 preferably is providedprophylactically to the patient, prior to, or concomitant with, thetherapy.

Below are several examples, describing protocols for demonstratingOP-3-induced tissue morphogenesis in bone, liver, nerve, dentin,cementum and periodontal tissue.

7.1 OP-3-Induced Bone Morphogenesis

A particularly useful mammalian tissue model system for demonstratingand evaluating the morphogenic activity of a protein is the endochondralbone tissue morphogenesis model known in the art and described, forexample, in U.S. Pat. No. 4,968,590 and incorporated herein byreference. The ability to induce endochondral bone formation includesthe ability to induce the proliferation of progenitor cells intochondroblasts and osteoblasts, the ability to induce cartilage matrixformation, cartilage calcification, and bone remodeling, and the abilityto induce formation of an appropriate vascular supply and hematopoeiticbone marrow differentiation.

The local environment in which the morphogenic material is placed isimportant for tissue morphogenesis. As used herein, "local environment"is understood to include the tissue structural matrix and theenvironment surrounding the tissue. For example, in addition to needingan appropriate anchoring substratum for their proliferation, the cellsstimulated by morphogens need signals to direct the tissue-specificityof their differentiation. These signals vary for the different tissuesand may include cell surface markers. In addition, vascularization ofnew tissue requires a local environment which'supports vascularization.

The following sets forth various procedures for evaluating the in vivomorphogenic utility of OP-3 and OP-3-containing compositions. Thecompositions may be injected or surgically implanted in a mammal,following any of a number of procedures well known in the art. Forexample, surgical implant bioassays may be performed essentiallyfollowing the procedure of Sampath et al. (1983) PNAS 80:6591-6595 andU.S. Pat. No. 4,968,590.

Histological sectioning and staining is preferred to determine theextent of morphogenesis in vivo, particularly in tissue repairprocedures. Excised implants are fixed in Bouins Solution, embedded inparaffin, and cut into 6-8 μm sections. Staining with toluidine blue orhemotoxylin/eosin demonstrates clearly the ultimate development of thenew tissue. Twelve day implants are usually sufficient to determinewhether the implants contain newly induced tissue.

Successful implants exhibit a controlled progression through the stagesof induced tissue development allowing one to identify and follow thetissue-specific events that occur. For example, in endochondral boneformation the stages include: (1) leukocytes on day one; (2) mesenchymalcell migration and proliferation on days two and three; (3) chondrocyteappearance on days five and six; (4) cartilage matrix formation on dayseven; (5) cartilage calcification on day eight; (6) vascular invasion,appearance of osteoblasts, and formation of new bone on days nine andten; (7) appearance of osteoclastic cells, and the commencement of boneremodeling and dissolution of the implanted matrix on days twelve toeighteen; and (8) hematopoietic bone marrow differentiation in theresulting ossicles on day twenty-one.

In addition to histological evaluation, biological markers may be usedas markers for tissue morphogenesis. Useful markers includetissue-specific enzymes whose activities may be assayed (e.g.,spectrophotometrically) after homogenization of the implant. Theseassays may be useful for quantitation and for rapidly obtaining anestimate of tissue formation after the implants are removed from theanimal. For example, alkaline phosphatase activity may be used as amarker for osteogenesis.

Incorporation of systemically provided OP-3 may be followed using taggedfragments (e.g., radioactively labelled) and determining theirlocalization in the new tissue, and/or by monitoring their disappearancefrom the circulatory system using a standard labeling protocol andpulse-chase procedure. OP-3 also may be provided with a tissue-specificmolecular tag, whose uptake may be monitored and correlated with theconcentration of OP-3 provided. As an example, ovary removal in femalerats results in reduced bone alkaline phosphatase activity, and rendersthe rats predisposed to osteoporosis (as described in Example 5). If thefemale rats now are provided with OP-3, a reduction in the systemicconcentration of calcium may be seen, which correlates with the presenceof the provided OP-3 and which is anticipated to correspond withincreased alkaline phosphatase activity.

7.2 Morphogen-Induced Liver Regeneration

As another example, a method for inducing morphogenesis of substantiallyinjured liver tissue following a partial hepatectomy utilizing OP-3 ispresented. Variations on this general protocol may be used to testmorphogen activity of OP-3 in other different tissues. The generalmethod involves excising an essentially nonregenerating portion of atissue and providing OP-3, preferably as a soluble pharmaceuticalpreparation to the excised tissue locus, closing the wound, andexamining the site at a future date. Like bone, liver has a potential toregenerate upon injury during post-fetal life.

OP-3, e.g., 1 mg/ml, in a biocompatible solution, for example, (e.g., apurified recombinant mature form of OP-3, is solubilized in 50% ethanol,or compatible solvent, containing 0.1% trifluoroacetic acid, orcompatible acid. Alternatively, the mature protein may be solubilized byassociation with a pro domain. The injectable OP-3 solution is prepared,e.g., by diluting one volume of OP-3 solvent-acid stock solution with 9volumes of 0.2% rat serum albumin in sterile PBS (phosphate-bufferedsaline).

In the experiment, growing rats or aged rats (e.g., Long Evans, CharlesRiver Laboratories, Wilmington) are anesthetized by using ketamine. Twoof the liver lobes (left and right) are cut out (approximately 1/3 ofthe lobe) and the OP-3 is injected locally at multiple sites along thecut ends. The amount of OP-3 injected may be, e.g., 100 μg in 1000 μl ofPBS/RSA (phosphate buffered saline/rat serum albumin) injection buffer.Placebo samples are injection buffer only. In experimental essays, fiverats in each group preferably are used. The wound is closed and the ratsare allowed to eat normal food and drink tap water.

After 12 days, the rats are sacrificed and liver regeneration isobserved visually, to evaluate the effects of the OP-3 on liverregeneration most effectively. The OP-3 fragment-injected group shows,e.g., complete liver tissue regeneration with no sign remaining of anycut in the liver. By contrast, the control group into which only PBS isinjected, shows only minimal regeneration with the incision remaining inthe sample. Previous experiments with other morphogens (e.g., OP-1) showthese morphogens alone induce liver tissue regeneration.

7.3 Morphogen-Induced Dentin, Cementum and Periodontal LigamentRegeneration

As still another example, the ability of OP-3 to induce dentinogenesisalso may be demonstrated. To date, the unpredictable response of dentalpulp tissue to injury is a basic clinical problem in dentistry.Cynomolgus monkeys are chosen as primate models as monkeys are presumedto be more indicative of human dental biology than models based on lowernon-primate mammals.

Using standard dental surgical procedures, small areas (e.g., 2 mm) ofdental pulps are surgically exposed by removing the enamel and dentinimmediately above the pulp (by drilling) of sample teeth, performing apartial amputation of the coronal pulp tissue, inducing hemostasis,application of the pulp treatment, and sealing and filling the cavity bystandard procedures.

Pulp treatments used may include: a morphogenically active fragment ofOP-3 dispersed in a carrier matrix; carrier matrix alone, and notreatment. Twelve teeth per animal (four for each treatment) areprepared, and two animals are used. At four weeks, teeth are extractedand processed histologically for analysis of dentin formation, and/orground to analyze dentin mineralization. The effect of OP-3 onosteodentin reparation may be observed visually by comparing controlsamples treatment (PBS) with OP-3. OP-3 plus a carrier matrix inducesformation of reparative or osteodentin bridges on surgically exposedhealthy dental pulps. By contrast, pulps treated with carrier matrixalone, do not form reparative dentin.

Similarly, implanting demineralized teeth and OP-3 into surgicallyprepared canine tooth sockets stimulates new periodontal tissueformation, including new cementum and periodontal ligament, as well asnew alveolar bone and dentin tissue, as described for OP-1 in U.S. Ser.No. 07/945,285, filed on even date herewith, abandoned, the disclosureof which is incorporated herein by reference. By contrast, untreatedteeth or teeth treated with carrier vehicle alone do not induceperiodontal tissue growth.

7.4 Morphogen-Induced Nerve Tissue Repair

As yet another example, the induction of regenerative effects on centralnervous system (CNS) repair, by a morphogenically active fragment ofOP-3, may be demonstrated using a rat brain stab model. Details of theprotocol are described in copending U.S. Ser. No. 922,813, abandoned.Briefly, male Long Evans rats are anesthetized and the head areaprepared for surgery. The calvariae is exposed using standard surgicalprocedures and a hole drilled toward the center of each lobe using a0.035K wire, just piercing the calvariae. 25 μl solutions containingeither morphogen (e.g., OP-3, 25 μg) or PBS then is provided to each ofthe holes by Hamilton syringe. Solutions are delivered to a depthapproximately 3 mm below the surface, into the underlying cortex, corpuscallosum and hippocampus. The skin then is sutured and the animalallowed to recover.

Three days post surgery, rats are sacrificed by decapitation and theirbrains processed for sectioning. Scar tissue formation is evaluated byimmunofluoresence staining for glial fibrillary acidic protein, a markerprotein for glial scarring, to qualitatively determine the degree ofscar formation. Sections also are probed with OP-3-specific antibody todetermine the presence of the protein. Reduced levels of glialfibrillary acidic protein are observed in the tissue sections of animalstreated with OP-3, evidencing the ability of the morphogen to inhibitglial scar formation, thereby stimulating nerve regeneration.

The ability of OP-3 to stimulate peripheral nervous system axonal growthover extended distances may be demonstrated using the following model.Neurons of the peripheral nervous system can sprout new processes ontheir own following injury, but without guidance these sproutingstypically fail to connect appropriately and die. Where the break isextensive, e.g., greater than 5 or 10 mm, regeneration is poor ornonexistent. Previous experiments with other morphogens, e.g., OP-1,show that morphogens stimulate peripheral nervous system axonal growthover extended distances, allowing repair and regeneration of damagedperipheral neural pathways.

In this example OP-3 stimulation of nerve regeneration is demonstratedusing the rat sciatic nerve model. The rat sciatic nerve can regeneratespontaneously across a 5 mm gap, and occasionally across a 10 mm gap,provided that the severed ends are inserted in a saline-filled nerveguidance channel. In this experiment, nerve regeneration across at leasta 12 mm gap is tested.

Adult female Sprague-Dawley rats (Charles River Laboratories, Inc.)weighing 230-250 g are anesthetized with intraperitoneal injections ofsodium pentobarbital (35 mg/kg body weight). A skin incision is madeparallel and just posterior to the femur. The avascular intermuscularplane between vastus lateralis and hamstring muscles are entered andfollowed to the loose fibroareolar tissue surrounding the sciatic nerve.The loose tissue is divided longitudinally thereby freeing the sciaticnerve over its full extent without devascularizing any portion. Under asurgical microscope the sciatic nerves are transected with-microscissors at mid-thigh and grafted with a OP-3 gel graft thatseparates the nerve stumps by 12 mm. The graft region is encased in asilicone tube 20 mm in length with a 1.5 mm inner diameter, the interiorof which is filled with the morphogen solution. Specifically, thecentral 12 mm of the tube consists of an OP-3 gel prepared by mixing 1to 5 μg of substantially pure recombinantly produced OP-3protein withapproximately 100 μl of MATRIGEL™ (from Collaborative Research, Inc.,Bedford, Mass.), an extracellular matrix extract derived from mousesarcoma tissue, and containing solubilized tissue basement membrane,including laminin, type IV collagen, heparin sulfate, proteoglycan andentactin, in phosphate-buffered saline. The morphogen-filled tube thenis implanted directly into the defect site, allowing 4 mm on each end toinsert the nerve stumps. Each stump is abutted against the morphogen geland is secured in the silicone tube by three stitches of commerciallyavailable surgical 10-0 nylon through the epineurium, the fascicleprotective sheath.

In addition to OP-3 gel grafts, control grafts of empty silicone tubes,silicone tubes filled with gel only and "reverse" autografts, wherein 12mm transected segments of the animal's sciatic nerve are rotated 180°prior to suturing, preferably also are grafted. All experimentspreferably are performed in quadruplicate. All wounds preferably areclosed by wound clips that are removed after 10 days. Rats can begrafted on both legs. At 3 weeks the animals are sacrificed, and thegrafted segments removed and frozen on dry ice immediately. Frozensections then are cut throughout the graft site, and examined for axonalregeneration by immunofluorescent staining using anti-neurofilamentantibodies labeled with flurocein (obtained, for example, from SigmaChemical Co., St. Louis).

Regeneration of the sciatic nerve occurs across the entire 12 mmdistance in all graft sites wherein the gap is filled with the OP-3 gel.By contrast, empty silicone tubes, gel alone and reverse autografts donot show nerve regeneration.

Example 8 Identification of Morphogen-Expressing Tissue

Determining the tissue distribution of morphogens may be used toidentify different morphogens expressed in a given tissue, as well as toidentify new, related morphogens. Tissue distribution also may be usedto identify useful morphogen-producing tissue for use in screening andidentifying candidate morphogen-stimulating agents. The morphogens (ortheir mRNA transcripts) readily are identified in different tissuesusing standard methodologies and minor modifications thereof in tissueswhere expression may be low. For example, protein distribution may bedetermined using standard Western blot analysis or immunofluorescenttechniques, and antibodies specific to the morphogen or morphogens ofinterest. Similarly, the distribution of morphogen transcripts may bedetermined using standard Northern hybridization protocols andtranscript-specific probes.

Any probe capable of hybridizing specifically to a transcript, anddistinguishing the transcript of interest from other, relatedtranscripts may be used. Because the morphogens described herein sharesuch high sequence homology in their active, C-terminal domains, thetissue distribution of a specific morphogen transcript may best bedetermined using a probe specific for the pro region of the immatureprotein and/or the N-terminal region of the mature protein. Anotheruseful sequence is the 3' non-coding region flanking and immediatelyfollowing the stop codon. These portions of the sequence varysubstantially among the morphogens of this invention, and accordingly,are specific for each protein. For example, a particularly usefulOP-3-specific probe sequence is one derived from a portion of the 3'untranslated sequence, e.g., nucleotides 1310-1674 of Seq. ID No. 1,which shares little or no homology with other morphogen sequences,including OP-2. The chosen fragment then is labelled using standardmeans well known and described in the art.

Using these morphogen-specific probes, which may be syntheticallyengineered or obtained from cloned sequences, morphogen transcripts canbe identified in mammalian tissue, using standard methodologies wellknown to those having ordinary skill in the art. A detailed descriptionof a suitable hybridization protocol is described in Ozkaynak, et al.,(1991) Biochem. Biophys. Res. Commn. 179:116-123, and Ozkaynak, et al.(1992) (JBC, in press), the disclosures of which are incorporated hereinby reference. Briefly, total RNA is prepared from various tissues (e.g.,murine embryo and developing and adult liver, kidney, testis, heart,brain, thymus, stomach) by a standard methodology such as by the methodof Chomczyaski et al. ((1987) Anal. Biochem 162:156-159) and describedbelow. Poly (A)+ RNA is prepared by using oligo (dT)-cellulosechromatography (e.g., Type 7, from Pharmacia LKB Biotechnology, Inc.).Poly (A)+ RNA (generally 15 μg) from each tissue is fractionated on a 1%agarose/formaldehyde gel and transferred onto a Nytran membrane(Schleicher & Schuell). Following the transfer, the membrane is baked at80° C. and the RNA is cross-linked under UV light (generally 30 secondsat 1 mW/cm²). Prior to hybridization, the appropriate probe is denaturedby heating. The hybridization is carried out in a lucite cylinderrotating in a roller bottle apparatus at approximately 1 rev/min forapproximately 15 hours at 37° C. using a hybridization mix of 40%formamide, 5×Denhardts, 5×SSPE, and 0.1% SDS. Following hybridization,the non-specific counts are washed off the filters in 0.1×SSPE, 0.1% SDSat 50° C.

An OP-3-specific 0.5 kb probe was made from a StuI-BglII fragment ofOP-3 cDNA. The fragment contains the 3' untranslated sequence fromnucleotides 1310-1674, plus an additional 140 bases. The fragment waslabelled using standard techniques and the hybridization performed asdescribed. To date, OP-3, like OP-2, appears to be expressed primarilyin early embryonic tissue. Specifically, Northern blots of murineembryos show abundant OP-3 expression in 8-day embryos, demonstrated bya strong band at 2.9 kb and a weaker band at 2.3 kb.

Example 9 Screening Assay for Candidate Compounds which Alter EndogenousMorphogen Levels

Candidate compound(s) which may be administered to affect the level ofendogenous OP-3 morphogen may be found using the following screeningassay, in which the level of OP-3 production by a cell type whichproduces measurable levels of the morphogen is determined with andwithout incubating the cell in culture with the compound, in order toassess the effects of the compound on the cell. This can be accomplishedby detection of the morphogen either at the protein or RNA level. Adetailed description also may be found in U.S. Ser. No. 752,861,abandoned, and U.S. Ser. No. 938,021, abandoned, incorporatedhereinabove by reference.

9.1 Growth of Cells in Culture

Cell cultures of kidney, adrenals, urinary bladder, brain, or otherorgans, may be prepared as described widely in the literature. Forexample, kidneys may be explanted from neonatal or new born or young oradult rodents (mouse or rat) and used in organ culture as whole orsliced (1-4 mm) tissues. Primary tissue cultures and established celllines, also derived from kidney, adrenals, urinary, bladder, brain,mammary, or other tissues may be established in multiwell plates (6 wellor 24 well) according to conventional cell culture techniques, and arecultured in the absence or presence of serum for a period of time (1-7days). Cells may be cultured, for example, in Dulbecco's Modified Eaglemedium (Gibco, Long Island, N.Y.) containing serum (e.g., fetal calfserum at 1%-10%, Gibco) or in serum-deprived medium, as desired, or indefined medium (e.g., containing insulin, transferrin, glucose, albumin,or other growth factors).

Samples for testing the level of morphogen production includes culturesupernatants or cell lysates, collected periodically and evaluated formorphogen production by immunoblot analysis (Sambrook et al., eds.,1989, Molecular Cloning, Cold Spring Harbor Press, Cold Spring Harbor,N.Y.), or a portion of the cell culture itself, collected periodicallyand used to prepare polyA+ RNA for RNA analysis. To monitor de novomorphogen synthesis, some cultures are labeled according to conventionalprocedures with an ³⁵ S-methionine/³⁵ S-cysteine mixture for 6-24 hoursand then evaluated for morphogenic protein synthesis by conventionalimmunoprecipitation methods.

9.2 Determination of Level of Morphogenic Protein

In order to quantitate the production of a morphogenic protein, e.g.,OP-3, by a cell type, an immunoassay may be performed to detect themorphogen using a polyclonal or monoclonal antibody specific for thatprotein. For example, OP-3 may be detected using a polyclonal antibodyspecific for OP-3 in an ELISA, as follows.

1 μg/100 μl of affinity-purified polyclonal rabbit IgG specific for OP-3is added to each well of a 96-well plate and incubated at 37° C. for anhour. The wells are washed four times with 0.167M sodium borate bufferwith 0.15M NaCl (BSB), pH 8.2, containing 0.1% Tween 20. To minimizenon-specific binding, the wells are blocked by filling completely with1% bovine serum albumin (BSA) in BSB and incubating for 1 hour at 37° C.The wells are then washed four times with BSB containing 0.1% Tween 20.A 100 μl aliquot of an appropriate dilution of each of the test samplesof cell culture supernatant is added to each well in triplicate andincubated at 37° C. for 30 min. After incubation, 100 μl biotinylatedrabbit anti-OP-3 serum (stock solution is about 1 mg/ml and diluted1:400 in BSB containing 1% BSA before use) is added to each well andincubated at 37° C. for 30 min. The wells are then washed four timeswith BSB containing 0.1% Tween 20. 100 μl strepavidin-alkaline (SouthernBiotechnology Associates, Inc. Birmingham, Ala., diluted 1:2000 in BSBcontaining 0.1% Tween 20 before use) is added to each well and incubatedat 37° C. for 30 min. The plates are washed four times with 0.5M Trisbuffered Saline (TBS), pH 7.2. 50 μl substrate (ELISA AmplificationSystem Kit, Life Technologies, Inc., Bethesda, Md.) is added to eachwell incubated at room temperature for 15 min. Then, 50 μl amplifier(from the same amplification system kit) is added and incubated foranother 15 min at room temperature. The reaction is stopped by theaddition of 50 μl 0.3M sulphuric acid. The OD at 490 nm of the solutionin each well is recorded. To quantitate OP-3 in culture media, an OP-3standard curve is performed in parallel with the test samples.

Polyclonal antibody may be prepared as follows. Each rabbit is given aprimary immunization of 100 ug/500 μl recombinantly-produced OP-3protein or protein fragment in 0.1% SDS mixed with 500 μl CompleteFreund's Adjuvant. The antigen is injected subcutaneously at multiplesites on the back and flanks of the animal. The rabbit is boosted aftera month in the same manner using incomplete Freund's Adjuvant. Testbleeds are taken from the ear vein seven days later. Two additionalboosts and test bleeds are performed at monthly intervals until antibodyagainst OP-3 is detected in the serum using an ELISA assay. Then, therabbit is boosted monthly with 100 μg of antigen and bled (15 ml perbleed) at days seven and ten after boosting.

Monoclonal antibody specific for a given morphogen may be prepared asfollows. A mouse is given two injections of OP-3 protein or a proteinfragment specific for OP-3. The protein preferably is recombinantlyproduced. The first injection contains 100 μg of OP-3 in completeFreund's adjuvant and is given subcutaneously. The second injectioncontains 50 μg of OP-3 in incomplete adjuvant and is givenintraperitoneally. The mouse then receives a total of 230 μg of OP-3 infour intraperitoneal injections at various times over an eight monthperiod. One week prior to fusion, the mouse is boosted intraperitoneallywith OP-3 (e.g., 100 μg) and may be additionally boosted with anOP-3-specific peptide (e.g., corresponding to the N-terminus of themature protein) conjugated to bovine serum albumin with a suitablecrosslinking agent. This boost can be repeated five days (IP), four days(IP), three days (IP) and one day (IV) prior to fusion. The mouse spleencells then are fused to commercially available myeloma cells at a ratioof 1:1 using PEG 1500 (Boeringer Mannheim, Germany), and the fused cellsplated and screened for OP-3-specific antibodies using OP-3 as antigen.The cell fusion and monoclonal screening steps readily are performedaccording to standard procedures well described in standard texts widelyavailable in the art.

Other Embodiments

The invention may be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof. The presentembodiments are therefore to be considered in all respects asillustrative and not restrictive, the scope of the invention beingindicated by the appended claims rather than by the foregoingdescription, and all changes which come within the meaning and range ofequivalency of the claims are therefore intended to be embraced therein.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 15                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1674 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE:                                                           (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 69..1265                                                        (D) OTHER INFORMATION: /note= "mOP3-PP"                                       (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GGATCCGCGGCGCTGTCCCATCCTTGTCGTCGAGGCGTCGCTGGATGCGAGTCCGCTAAA60                CGTCCGAGATGGCTGCGCGTCCGGGACTCCTATGGCTACTGGGCCTGGCT110                         MetAlaAlaArgProGlyLeuLeuTrpLeuLeuGlyLeuAla                                    1510                                                                          CTGTGCGTGTTGGGCGGCGGTCACCTCTCGCATCCCCCGCACGTCTTT158                           LeuCysValLeuGlyGlyGlyHisLeuSerHisProProHisValPhe                              15202530                                                                      CCCCAGCGTCGACTAGGAGTACGCGAGCCCCGCGACATGCAGCGCGAG206                           ProGlnArgArgLeuGlyValArgGluProArgAspMetGlnArgGlu                              354045                                                                        ATTCGGGAGGTGCTGGGGCTAGCCGGGCGGCCCCGATCCCGAGCACCG254                           IleArgGluValLeuGlyLeuAlaGlyArgProArgSerArgAlaPro                              505560                                                                        GTCGGGGCTGCCCAGCAGCCAGCGTCTGCGCCCCTCTTTATGTTGGAC302                           ValGlyAlaAlaGlnGlnProAlaSerAlaProLeuPheMetLeuAsp                              657075                                                                        CTGTACCGTGCCATGACGGATGACAGTGGCGGTGGGACCCCGCAGCCT350                           LeuTyrArgAlaMetThrAspAspSerGlyGlyGlyThrProGlnPro                              808590                                                                        CACTTGGACCGTGCTGACCTGATTATGAGCTTTGTCAACATAGTGGAA398                           HisLeuAspArgAlaAspLeuIleMetSerPheValAsnIleValGlu                              95100105110                                                                   CGCGACCGTACCCTGGGCTACCAGGAGCCACACTGGAAGGAATTCCAC446                           ArgAspArgThrLeuGlyTyrGlnGluProHisTrpLysGluPheHis                              115120125                                                                     TTTGACCTAACCCAGATCCCTGCTGGGGAGGCTGTCACAGCTGCTGAG494                           PheAspLeuThrGlnIleProAlaGlyGluAlaValThrAlaAlaGlu                              130135140                                                                     TTCCGGATCTACAAAGAACCCAGTACCCACCCGCTCAACACAACCCTC542                           PheArgIleTyrLysGluProSerThrHisProLeuAsnThrThrLeu                              145150155                                                                     CACATCAGCATGTTCGAAGTGGTCCAAGAGCACTCCAACAGGGAGTCT590                           HisIleSerMetPheGluValValGlnGluHisSerAsnArgGluSer                              160165170                                                                     GACTTGTTCTTTTTGGATCTTCAGACGCTCCGATCTGGGGACGAGGGC638                           AspLeuPhePheLeuAspLeuGlnThrLeuArgSerGlyAspGluGly                              175180185190                                                                  TGGCTGGTGCTGGACATCACAGCAGCCAGTGACCGATGGCTGCTGAAC686                           TrpLeuValLeuAspIleThrAlaAlaSerAspArgTrpLeuLeuAsn                              195200205                                                                     CATCACAAGGACCTAGGACTCCGCCTCTATGTGGAAACCGAGGATGGG734                           HisHisLysAspLeuGlyLeuArgLeuTyrValGluThrGluAspGly                              210215220                                                                     CACAGCATAGATCCTGGCCTAGCTGGTCTGCTTGGACGACAAGCACCA782                           HisSerIleAspProGlyLeuAlaGlyLeuLeuGlyArgGlnAlaPro                              225230235                                                                     CGCTCCAGACAGCCTTTCATGGTTGGTTTCTTCAGGGCCAACCAGAGT830                           ArgSerArgGlnProPheMetValGlyPhePheArgAlaAsnGlnSer                              240245250                                                                     CCTGTGCGGGCCCCTCGAACAGCAAGACCACTGAAGAAGAAGCAGCTA878                           ProValArgAlaProArgThrAlaArgProLeuLysLysLysGlnLeu                              255260265270                                                                  AATCAAATCAACCAGCTGCCGCACTCCAACAAACACCTAGGAATCCTT926                           AsnGlnIleAsnGlnLeuProHisSerAsnLysHisLeuGlyIleLeu                              275280285                                                                     GATGATGGCCACGGTTCTCACGGCAGAGAAGTTTGCCGCAGGCATGAG974                           AspAspGlyHisGlySerHisGlyArgGluValCysArgArgHisGlu                              290295300                                                                     CTCTATGTCAGCTTCCGTGACCTTGGCTGGCTGGACTCTGTCATTGCC1022                          LeuTyrValSerPheArgAspLeuGlyTrpLeuAspSerValIleAla                              305310315                                                                     CCCCAGGGCTACTCCGCCTATTACTGTGCTGGGGAGTGCATCTACCCA1070                          ProGlnGlyTyrSerAlaTyrTyrCysAlaGlyGluCysIleTyrPro                              320325330                                                                     CTGAACTCCTGTATGAACTCCACCAACCACGCCACTATGCAGGCCCTG1118                          LeuAsnSerCysMetAsnSerThrAsnHisAlaThrMetGlnAlaLeu                              335340345350                                                                  GTACATCTGATGAAGCCAGATATCATCCCCAAGGTGTGCTGTGTGCCT1166                          ValHisLeuMetLysProAspIleIleProLysValCysCysValPro                              355360365                                                                     ACTGAGCTGAGTGCCATTTCTCTGCTCTACTATGATAGAAACAATAAT1214                          ThrGluLeuSerAlaIleSerLeuLeuTyrTyrAspArgAsnAsnAsn                              370375380                                                                     GTCATCCTGCGCAGGGAGCGCAACATGGTAGTCCAGGCCTGTGGCTGC1262                          ValIleLeuArgArgGluArgAsnMetValValGlnAlaCysGlyCys                              385390395                                                                     CACTGAGTCCCTGCCCAACAGCCTGCTGCCATCCCATCTATCTAGTCAGGCCT1315                     His                                                                           CTCTTCCAAGGCAGGAAACCAACAAAGAGGGAAGGCAGTGCTTTCAACTCCATGTCCACA1375              TTCACAGTCTTGGCCCTCTCTGTTCTTTTTGCCAAGGCTGAGAAGATGGTCCTAGTTATA1435              ACCCTGGTGACCTCAGTAGCCCGATCTCTCATCTCCCCAAACTCCCCAATGCAGCCAGGG1495              GCATCTATGTCCTTTGGGATTGGGCACAGAAGTCCAATTTACCAACTTATTCATGAGTCA1555              CTACTGGCCCAGCCTGGACTTGAACCTGGAACACAGGGTAGAGCTCAGGCTCTTCAGTAT1615              CCATCAGAAGATTTAGGTGTGTGCAGACATGACCACACTCCCCCTAGCACTCCATAGCC1674               (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 399 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       MetAlaAlaArgProGlyLeuLeuTrpLeuLeuGlyLeuAlaLeuCys                              151015                                                                        ValLeuGlyGlyGlyHisLeuSerHisProProHisValPheProGln                              202530                                                                        ArgArgLeuGlyValArgGluProArgAspMetGlnArgGluIleArg                              354045                                                                        GluValLeuGlyLeuAlaGlyArgProArgSerArgAlaProValGly                              505560                                                                        AlaAlaGlnGlnProAlaSerAlaProLeuPheMetLeuAspLeuTyr                              65707580                                                                      ArgAlaMetThrAspAspSerGlyGlyGlyThrProGlnProHisLeu                              859095                                                                        AspArgAlaAspLeuIleMetSerPheValAsnIleValGluArgAsp                              100105110                                                                     ArgThrLeuGlyTyrGlnGluProHisTrpLysGluPheHisPheAsp                              115120125                                                                     LeuThrGlnIleProAlaGlyGluAlaValThrAlaAlaGluPheArg                              130135140                                                                     IleTyrLysGluProSerThrHisProLeuAsnThrThrLeuHisIle                              145150155160                                                                  SerMetPheGluValValGlnGluHisSerAsnArgGluSerAspLeu                              165170175                                                                     PhePheLeuAspLeuGlnThrLeuArgSerGlyAspGluGlyTrpLeu                              180185190                                                                     ValLeuAspIleThrAlaAlaSerAspArgTrpLeuLeuAsnHisHis                              195200205                                                                     LysAspLeuGlyLeuArgLeuTyrValGluThrGluAspGlyHisSer                              210215220                                                                     IleAspProGlyLeuAlaGlyLeuLeuGlyArgGlnAlaProArgSer                              225230235240                                                                  ArgGlnProPheMetValGlyPhePheArgAlaAsnGlnSerProVal                              245250255                                                                     ArgAlaProArgThrAlaArgProLeuLysLysLysGlnLeuAsnGln                              260265270                                                                     IleAsnGlnLeuProHisSerAsnLysHisLeuGlyIleLeuAspAsp                              275280285                                                                     GlyHisGlySerHisGlyArgGluValCysArgArgHisGluLeuTyr                              290295300                                                                     ValSerPheArgAspLeuGlyTrpLeuAspSerValIleAlaProGln                              305310315320                                                                  GlyTyrSerAlaTyrTyrCysAlaGlyGluCysIleTyrProLeuAsn                              325330335                                                                     SerCysMetAsnSerThrAsnHisAlaThrMetGlnAlaLeuValHis                              340345350                                                                     LeuMetLysProAspIleIleProLysValCysCysValProThrGlu                              355360365                                                                     LeuSerAlaIleSerLeuLeuTyrTyrAspArgAsnAsnAsnValIle                              370375380                                                                     LeuArgArgGluArgAsnMetValValGlnAlaCysGlyCysHis                                 385390395                                                                     (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1822 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 49..1341                                                        (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"                         /product= "hOP1-PP"                                                           /note= "hOP1 cDNA"                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       GGTGCGGGCCCGGAGCCCGGAGCCCGGGTAGCGCGTAGAGCCGGCGCGATGCACGTG57                   MetHisVal                                                                     CGCTCACTGCGAGCTGCGGCGCCGCACAGCTTCGTGGCGCTCTGGGCA105                           ArgSerLeuArgAlaAlaAlaProHisSerPheValAlaLeuTrpAla                              51015                                                                         CCCCTGTTCCTGCTGCGCTCCGCCCTGGCCGACTTCAGCCTGGACAAC153                           ProLeuPheLeuLeuArgSerAlaLeuAlaAspPheSerLeuAspAsn                              20253035                                                                      GAGGTGCACTCGAGCTTCATCCACCGGCGCCTCCGCAGCCAGGAGCGG201                           GluValHisSerSerPheIleHisArgArgLeuArgSerGlnGluArg                              404550                                                                        CGGGAGATGCAGCGCGAGATCCTCTCCATTTTGGGCTTGCCCCACCGC249                           ArgGluMetGlnArgGluIleLeuSerIleLeuGlyLeuProHisArg                              556065                                                                        CCGCGCCCGCACCTCCAGGGCAAGCACAACTCGGCACCCATGTTCATG297                           ProArgProHisLeuGlnGlyLysHisAsnSerAlaProMetPheMet                              707580                                                                        CTGGACCTGTACAACGCCATGGCGGTGGAGGAGGGCGGCGGGCCCGGC345                           LeuAspLeuTyrAsnAlaMetAlaValGluGluGlyGlyGlyProGly                              859095                                                                        GGCCAGGGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGC393                           GlyGlnGlyPheSerTyrProTyrLysAlaValPheSerThrGlnGly                              100105110115                                                                  CCCCCTCTGGCCAGCCTGCAAGATAGCCATTTCCTCACCGACGCCGAC441                           ProProLeuAlaSerLeuGlnAspSerHisPheLeuThrAspAlaAsp                              120125130                                                                     ATGGTCATGAGCTTCGTCAACCTCGTGGAACATGACAAGGAATTCTTC489                           MetValMetSerPheValAsnLeuValGluHisAspLysGluPhePhe                              135140145                                                                     CACCCACGCTACCACCATCGAGAGTTCCGGTTTGATCTTTCCAAGATC537                           HisProArgTyrHisHisArgGluPheArgPheAspLeuSerLysIle                              150155160                                                                     CCAGAAGGGGAAGCTGTCACGGCAGCCGAATTCCGGATCTACAAGGAC585                           ProGluGlyGluAlaValThrAlaAlaGluPheArgIleTyrLysAsp                              165170175                                                                     TACATCCGGGAACGCTTCGACAATGAGACGTTCCGGATCAGCGTTTAT633                           TyrIleArgGluArgPheAspAsnGluThrPheArgIleSerValTyr                              180185190195                                                                  CAGGTGCTCCAGGAGCACTTGGGCAGGGAATCGGATCTCTTCCTGCTC681                           GlnValLeuGlnGluHisLeuGlyArgGluSerAspLeuPheLeuLeu                              200205210                                                                     GACAGCCGTACCCTCTGGGCCTCGGAGGAGGGCTGGCTGGTGTTTGAC729                           AspSerArgThrLeuTrpAlaSerGluGluGlyTrpLeuValPheAsp                              215220225                                                                     ATCACAGCCACCAGCAACCACTGGGTGGTCAATCCGCGGCACAACCTG777                           IleThrAlaThrSerAsnHisTrpValValAsnProArgHisAsnLeu                              230235240                                                                     GGCCTGCAGCTCTCGGTGGAGACGCTGGATGGGCAGAGCATCAACCCC825                           GlyLeuGlnLeuSerValGluThrLeuAspGlyGlnSerIleAsnPro                              245250255                                                                     AAGTTGGCGGGCCTGATTGGGCGGCACGGGCCCCAGAACAAGCAGCCC873                           LysLeuAlaGlyLeuIleGlyArgHisGlyProGlnAsnLysGlnPro                              260265270275                                                                  TTCATGGTGGCTTTCTTCAAGGCCACGGAGGTCCACTTCCGCAGCATC921                           PheMetValAlaPhePheLysAlaThrGluValHisPheArgSerIle                              280285290                                                                     CGGTCCACGGGGAGCAAACAGCGCAGCCAGAACCGCTCCAAGACGCCC969                           ArgSerThrGlySerLysGlnArgSerGlnAsnArgSerLysThrPro                              295300305                                                                     AAGAACCAGGAAGCCCTGCGGATGGCCAACGTGGCAGAGAACAGCAGC1017                          LysAsnGlnGluAlaLeuArgMetAlaAsnValAlaGluAsnSerSer                              310315320                                                                     AGCGACCAGAGGCAGGCCTGTAAGAAGCACGAGCTGTATGTCAGCTTC1065                          SerAspGlnArgGlnAlaCysLysLysHisGluLeuTyrValSerPhe                              325330335                                                                     CGAGACCTGGGCTGGCAGGACTGGATCATCGCGCCTGAAGGCTACGCC1113                          ArgAspLeuGlyTrpGlnAspTrpIleIleAlaProGluGlyTyrAla                              340345350355                                                                  GCCTACTACTGTGAGGGGGAGTGTGCCTTCCCTCTGAACTCCTACATG1161                          AlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsnSerTyrMet                              360365370                                                                     AACGCCACCAACCACGCCATCGTGCAGACGCTGGTCCACTTCATCAAC1209                          AsnAlaThrAsnHisAlaIleValGlnThrLeuValHisPheIleAsn                              375380385                                                                     CCGGAAACGGTGCCCAAGCCCTGCTGTGCGCCCACGCAGCTCAATGCC1257                          ProGluThrValProLysProCysCysAlaProThrGlnLeuAsnAla                              390395400                                                                     ATCTCCGTCCTCTACTTCGATGACAGCTCCAACGTCATCCTGAAGAAA1305                          IleSerValLeuTyrPheAspAspSerSerAsnValIleLeuLysLys                              405410415                                                                     TACAGAAACATGGTGGTCCGGGCCTGTGGCTGCCACTAGCTCCTCC1351                            TyrArgAsnMetValValArgAlaCysGlyCysHis                                          420425430                                                                     GAGAATTCAGACCCTTTGGGGCCAAGTTTTTCTGGATCCTCCATTGCTCGCCTTGGCCAG1411              GAACCAGCAGACCAACTGCCTTTTGTGAGACCTTCCCCTCCCTATCCCCAACTTTAAAGG1471              TGTGAGAGTATTAGGAAACATGAGCAGCATATGGCTTTTGATCAGTTTTTCAGTGGCAGC1531              ATCCAATGAACAAGATCCTACAAGCTGTGCAGGCAAAACCTAGCAGGAAAAAAAAACAAC1591              GCATAAAGAAAAATGGCCGGGCCAGGTCATTGGCTGGGAAGTCTCAGCCATGCACGGACT1651              CGTTTCCAGAGGTAATTATGAGCGCCTACCAGCCAGGCCACCCAGCCGTGGGAGGAAGGG1711              GGCGTGGCAAGGGGTGGGCACATTGGTGTCTGTGCGAAAGGAAAATTGACCCGGAAGTTC1771              CTGTAATAAATGTCACAATAAAACGAATGAATGAAAAAAAAAAAAAAAAAA1822                       (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 431 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       MetHisValArgSerLeuArgAlaAlaAlaProHisSerPheValAla                              151015                                                                        LeuTrpAlaProLeuPheLeuLeuArgSerAlaLeuAlaAspPheSer                              202530                                                                        LeuAspAsnGluValHisSerSerPheIleHisArgArgLeuArgSer                              354045                                                                        GlnGluArgArgGluMetGlnArgGluIleLeuSerIleLeuGlyLeu                              505560                                                                        ProHisArgProArgProHisLeuGlnGlyLysHisAsnSerAlaPro                              65707580                                                                      MetPheMetLeuAspLeuTyrAsnAlaMetAlaValGluGluGlyGly                              859095                                                                        GlyProGlyGlyGlnGlyPheSerTyrProTyrLysAlaValPheSer                              100105110                                                                     ThrGlnGlyProProLeuAlaSerLeuGlnAspSerHisPheLeuThr                              115120125                                                                     AspAlaAspMetValMetSerPheValAsnLeuValGluHisAspLys                              130135140                                                                     GluPhePheHisProArgTyrHisHisArgGluPheArgPheAspLeu                              145150155160                                                                  SerLysIleProGluGlyGluAlaValThrAlaAlaGluPheArgIle                              165170175                                                                     TyrLysAspTyrIleArgGluArgPheAspAsnGluThrPheArgIle                              180185190                                                                     SerValTyrGlnValLeuGlnGluHisLeuGlyArgGluSerAspLeu                              195200205                                                                     PheLeuLeuAspSerArgThrLeuTrpAlaSerGluGluGlyTrpLeu                              210215220                                                                     ValPheAspIleThrAlaThrSerAsnHisTrpValValAsnProArg                              225230235240                                                                  HisAsnLeuGlyLeuGlnLeuSerValGluThrLeuAspGlyGlnSer                              245250255                                                                     IleAsnProLysLeuAlaGlyLeuIleGlyArgHisGlyProGlnAsn                              260265270                                                                     LysGlnProPheMetValAlaPhePheLysAlaThrGluValHisPhe                              275280285                                                                     ArgSerIleArgSerThrGlySerLysGlnArgSerGlnAsnArgSer                              290295300                                                                     LysThrProLysAsnGlnGluAlaLeuArgMetAlaAsnValAlaGlu                              305310315320                                                                  AsnSerSerSerAspGlnArgGlnAlaCysLysLysHisGluLeuTyr                              325330335                                                                     ValSerPheArgAspLeuGlyTrpGlnAspTrpIleIleAlaProGlu                              340345350                                                                     GlyTyrAlaAlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsn                              355360365                                                                     SerTyrMetAsnAlaThrAsnHisAlaIleValGlnThrLeuValHis                              370375380                                                                     PheIleAsnProGluThrValProLysProCysCysAlaProThrGln                              385390395400                                                                  LeuAsnAlaIleSerValLeuTyrPheAspAspSerSerAsnValIle                              405410415                                                                     LeuLysLysTyrArgAsnMetValValArgAlaCysGlyCysHis                                 420425430                                                                     (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1873 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 104..1393                                                       (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"                         /product= "mOP1-PP"                                                           /note= "mOP1 cDNA"                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       CTGCAGCAAGTGACCTCGGGTCGTGGACCGCTGCCCTGCCCCCTCCGCTGCCACCTGGGG60                CGGCGCGGGCCCGGTGCCCCGGATCGCGCGTAGAGCCGGCGCGATGCACGTGCGC115                    MetHisValArg                                                                  1                                                                             TCGCTGCGCGCTGCGGCGCCACACAGCTTCGTGGCGCTCTGGGCGCCT163                           SerLeuArgAlaAlaAlaProHisSerPheValAlaLeuTrpAlaPro                              5101520                                                                       CTGTTCTTGCTGCGCTCCGCCCTGGCCGATTTCAGCCTGGACAACGAG211                           LeuPheLeuLeuArgSerAlaLeuAlaAspPheSerLeuAspAsnGlu                              253035                                                                        GTGCACTCCAGCTTCATCCACCGGCGCCTCCGCAGCCAGGAGCGGCGG259                           ValHisSerSerPheIleHisArgArgLeuArgSerGlnGluArgArg                              404550                                                                        GAGATGCAGCGGGAGATCCTGTCCATCTTAGGGTTGCCCCATCGCCCG307                           GluMetGlnArgGluIleLeuSerIleLeuGlyLeuProHisArgPro                              556065                                                                        CGCCCGCACCTCCAGGGAAAGCATAATTCGGCGCCCATGTTCATGTTG355                           ArgProHisLeuGlnGlyLysHisAsnSerAlaProMetPheMetLeu                              707580                                                                        GACCTGTACAACGCCATGGCGGTGGAGGAGAGCGGGCCGGACGGACAG403                           AspLeuTyrAsnAlaMetAlaValGluGluSerGlyProAspGlyGln                              859095100                                                                     GGCTTCTCCTACCCCTACAAGGCCGTCTTCAGTACCCAGGGCCCCCCT451                           GlyPheSerTyrProTyrLysAlaValPheSerThrGlnGlyProPro                              105110115                                                                     TTAGCCAGCCTGCAGGACAGCCATTTCCTCACTGACGCCGACATGGTC499                           LeuAlaSerLeuGlnAspSerHisPheLeuThrAspAlaAspMetVal                              120125130                                                                     ATGAGCTTCGTCAACCTAGTGGAACATGACAAAGAATTCTTCCACCCT547                           MetSerPheValAsnLeuValGluHisAspLysGluPhePheHisPro                              135140145                                                                     CGATACCACCATCGGGAGTTCCGGTTTGATCTTTCCAAGATCCCCGAG595                           ArgTyrHisHisArgGluPheArgPheAspLeuSerLysIleProGlu                              150155160                                                                     GGCGAACGGGTGACCGCAGCCGAATTCAGGATCTATAAGGACTACATC643                           GlyGluArgValThrAlaAlaGluPheArgIleTyrLysAspTyrIle                              165170175180                                                                  CGGGAGCGATTTGACAACGAGACCTTCCAGATCACAGTCTATCAGGTG691                           ArgGluArgPheAspAsnGluThrPheGlnIleThrValTyrGlnVal                              185190195                                                                     CTCCAGGAGCACTCAGGCAGGGAGTCGGACCTCTTCTTGCTGGACAGC739                           LeuGlnGluHisSerGlyArgGluSerAspLeuPheLeuLeuAspSer                              200205210                                                                     CGCACCATCTGGGCTTCTGAGGAGGGCTGGTTGGTGTTTGATATCACA787                           ArgThrIleTrpAlaSerGluGluGlyTrpLeuValPheAspIleThr                              215220225                                                                     GCCACCAGCAACCACTGGGTGGTCAACCCTCGGCACAACCTGGGCTTA835                           AlaThrSerAsnHisTrpValValAsnProArgHisAsnLeuGlyLeu                              230235240                                                                     CAGCTCTCTGTGGAGACCCTGGATGGGCAGAGCATCAACCCCAAGTTG883                           GlnLeuSerValGluThrLeuAspGlyGlnSerIleAsnProLysLeu                              245250255260                                                                  GCAGGCCTGATTGGACGGCATGGACCCCAGAACAAGCAACCCTTCATG931                           AlaGlyLeuIleGlyArgHisGlyProGlnAsnLysGlnProPheMet                              265270275                                                                     GTGGCCTTCTTCAAGGCCACGGAAGTCCATCTCCGTAGTATCCGGTCC979                           ValAlaPhePheLysAlaThrGluValHisLeuArgSerIleArgSer                              280285290                                                                     ACGGGGGGCAAGCAGCGCAGCCAGAATCGCTCCAAGACGCCAAAGAAC1027                          ThrGlyGlyLysGlnArgSerGlnAsnArgSerLysThrProLysAsn                              295300305                                                                     CAAGAGGCCCTGAGGATGGCCAGTGTGGCAGAAAACAGCAGCAGTGAC1075                          GlnGluAlaLeuArgMetAlaSerValAlaGluAsnSerSerSerAsp                              310315320                                                                     CAGAGGCAGGCCTGCAAGAAACATGAGCTGTACGTCAGCTTCCGAGAC1123                          GlnArgGlnAlaCysLysLysHisGluLeuTyrValSerPheArgAsp                              325330335340                                                                  CTTGGCTGGCAGGACTGGATCATTGCACCTGAAGGCTATGCTGCCTAC1171                          LeuGlyTrpGlnAspTrpIleIleAlaProGluGlyTyrAlaAlaTyr                              345350355                                                                     TACTGTGAGGGAGAGTGCGCCTTCCCTCTGAACTCCTACATGAACGCC1219                          TyrCysGluGlyGluCysAlaPheProLeuAsnSerTyrMetAsnAla                              360365370                                                                     ACCAACCACGCCATCGTCCAGACACTGGTTCACTTCATCAACCCAGAC1267                          ThrAsnHisAlaIleValGlnThrLeuValHisPheIleAsnProAsp                              375380385                                                                     ACAGTACCCAAGCCCTGCTGTGCGCCCACCCAGCTCAACGCCATCTCT1315                          ThrValProLysProCysCysAlaProThrGlnLeuAsnAlaIleSer                              390395400                                                                     GTCCTCTACTTCGACGACAGCTCTAATGTCATCCTGAAGAAGTACAGA1363                          ValLeuTyrPheAspAspSerSerAsnValIleLeuLysLysTyrArg                              405410415420                                                                  AACATGGTGGTCCGGGCCTGTGGCTGCCACTAGCTCTTCCTGAGACCCTG1413                        AsnMetValValArgAlaCysGlyCysHis                                                425430                                                                        ACCTTTGCGGGGCCACACCTTTCCAAATCTTCGATGTCTCACCATCTAAGTCTCTCACTG1473              CCCACCTTGGCGAGGAGAACAGACCAACCTCTCCTGAGCCTTCCCTCACCTCCCAACCGG1533              AAGCATGTAAGGGTTCCAGAAACCTGAGCGTGCAGCAGCTGATGAGCGCCCTTTCCTTCT1593              GGCACGTGACGGACAAGATCCTACCAGCTACCACAGCAAACGCCTAAGAGCAGGAAAAAT1653              GTCTGCCAGGAAAGTGTCCAGTGTCCACATGGCCCCTGGCGCTCTGAGTCTTTGAGGAGT1713              AATCGCAAGCCTCGTTCAGCTGCAGCAGAAGGAAGGGCTTAGCCAGGGTGGGCGCTGGCG1773              TCTGTGTTGAAGGGAAACCAAGCAGAAGCCACTGTAATGATATGTCACAATAAAACCCAT1833              GAATGAAAAAAAAAAAAAAAAAAAAAAAAAAAAAGAATTC1873                                  (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 430 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       MetHisValArgSerLeuArgAlaAlaAlaProHisSerPheValAla                              151015                                                                        LeuTrpAlaProLeuPheLeuLeuArgSerAlaLeuAlaAspPheSer                              202530                                                                        LeuAspAsnGluValHisSerSerPheIleHisArgArgLeuArgSer                              354045                                                                        GlnGluArgArgGluMetGlnArgGluIleLeuSerIleLeuGlyLeu                              505560                                                                        ProHisArgProArgProHisLeuGlnGlyLysHisAsnSerAlaPro                              65707580                                                                      MetPheMetLeuAspLeuTyrAsnAlaMetAlaValGluGluSerGly                              859095                                                                        ProAspGlyGlnGlyPheSerTyrProTyrLysAlaValPheSerThr                              100105110                                                                     GlnGlyProProLeuAlaSerLeuGlnAspSerHisPheLeuThrAsp                              115120125                                                                     AlaAspMetValMetSerPheValAsnLeuValGluHisAspLysGlu                              130135140                                                                     PhePheHisProArgTyrHisHisArgGluPheArgPheAspLeuSer                              145150155160                                                                  LysIleProGluGlyGluArgValThrAlaAlaGluPheArgIleTyr                              165170175                                                                     LysAspTyrIleArgGluArgPheAspAsnGluThrPheGlnIleThr                              180185190                                                                     ValTyrGlnValLeuGlnGluHisSerGlyArgGluSerAspLeuPhe                              195200205                                                                     LeuLeuAspSerArgThrIleTrpAlaSerGluGluGlyTrpLeuVal                              210215220                                                                     PheAspIleThrAlaThrSerAsnHisTrpValValAsnProArgHis                              225230235240                                                                  AsnLeuGlyLeuGlnLeuSerValGluThrLeuAspGlyGlnSerIle                              245250255                                                                     AsnProLysLeuAlaGlyLeuIleGlyArgHisGlyProGlnAsnLys                              260265270                                                                     GlnProPheMetValAlaPhePheLysAlaThrGluValHisLeuArg                              275280285                                                                     SerIleArgSerThrGlyGlyLysGlnArgSerGlnAsnArgSerLys                              290295300                                                                     ThrProLysAsnGlnGluAlaLeuArgMetAlaSerValAlaGluAsn                              305310315320                                                                  SerSerSerAspGlnArgGlnAlaCysLysLysHisGluLeuTyrVal                              325330335                                                                     SerPheArgAspLeuGlyTrpGlnAspTrpIleIleAlaProGluGly                              340345350                                                                     TyrAlaAlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsnSer                              355360365                                                                     TyrMetAsnAlaThrAsnHisAlaIleValGlnThrLeuValHisPhe                              370375380                                                                     IleAsnProAspThrValProLysProCysCysAlaProThrGlnLeu                              385390395400                                                                  AsnAlaIleSerValLeuTyrPheAspAspSerSerAsnValIleLeu                              405410415                                                                     LysLysTyrArgAsnMetValValArgAlaCysGlyCysHis                                    420425430                                                                     (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1723 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 490..1696                                                       (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"                         /product= "hOP2-PP"                                                           /note= "hOP2 cDNA"                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GGCGCCGGCAGAGCAGGAGTGGCTGGAGGAGCTGTGGTTGGAGCAGGAGGTGGCACGGCA60                GGGCTGGAGGGCTCCCTATGAGTGGCGGAGACGGCCCAGGAGGCGCTGGAGCAACAGCTC120               CCACACCGCACCAAGCGGTGGCTGCAGGAGCTCGCCCATCGCCCCTGCGCTGCTCGGACC180               GCGGCCACAGCCGGACTGGCGGGTACGGCGGCGACAGAGGCATTGGCCGAGAGTCCCAGT240               CCGCAGAGTAGCCCCGGCCTCGAGGCGGTGGCGTCCCGGTCCTCTCCGTCCAGGAGCCAG300               GACAGGTGTCGCGCGGCGGGGCTCCAGGGACCGCGCCTGAGGCCGGCTGCCCGCCCGTCC360               CGCCCCGCCCCGCCGCCCGCCGCCCGCCGAGCCCAGCCTCCTTGCCGTCGGGGCGTCCCC420               AGGCCCTGGGTCGGCCGCGGAGCCGATGCGCGCCCGCTGAGCGCCCCAGCTGAGCGCCCC480               CGGCCTGCCATGACCGCGCTCCCCGGCCCGCTCTGGCTCCTGGGCCTG528                           MetThrAlaLeuProGlyProLeuTrpLeuLeuGlyLeu                                       1510                                                                          GCGCTATGCGCGCTGGGCGGGGGCGGCCCCGGCCTGCGACCCCCGCCC576                           AlaLeuCysAlaLeuGlyGlyGlyGlyProGlyLeuArgProProPro                              152025                                                                        GGCTGTCCCCAGCGACGTCTGGGCGCGCGCGAGCGCCGGGACGTGCAG624                           GlyCysProGlnArgArgLeuGlyAlaArgGluArgArgAspValGln                              30354045                                                                      CGCGAGATCCTGGCGGTGCTCGGGCTGCCTGGGCGGCCCCGGCCCCGC672                           ArgGluIleLeuAlaValLeuGlyLeuProGlyArgProArgProArg                              505560                                                                        GCGCCACCCGCCGCCTCCCGGCTGCCCGCGTCCGCGCCGCTCTTCATG720                           AlaProProAlaAlaSerArgLeuProAlaSerAlaProLeuPheMet                              657075                                                                        CTGGACCTGTACCACGCCATGGCCGGCGACGACGACGAGGACGGCGCG768                           LeuAspLeuTyrHisAlaMetAlaGlyAspAspAspGluAspGlyAla                              808590                                                                        CCCGCGGAGCGGCGCCTGGGCCGCGCCGACCTGGTCATGAGCTTCGTT816                           ProAlaGluArgArgLeuGlyArgAlaAspLeuValMetSerPheVal                              95100105                                                                      AACATGGTGGAGCGAGACCGTGCCCTGGGCCACCAGGAGCCCCATTGG864                           AsnMetValGluArgAspArgAlaLeuGlyHisGlnGluProHisTrp                              110115120125                                                                  AAGGAGTTCCGCTTTGACCTGACCCAGATCCCGGCTGGGGAGGCGGTC912                           LysGluPheArgPheAspLeuThrGlnIleProAlaGlyGluAlaVal                              130135140                                                                     ACAGCTGCGGAGTTCCGGATTTACAAGGTGCCCAGCATCCACCTGCTC960                           ThrAlaAlaGluPheArgIleTyrLysValProSerIleHisLeuLeu                              145150155                                                                     AACAGGACCCTCCACGTCAGCATGTTCCAGGTGGTCCAGGAGCAGTCC1008                          AsnArgThrLeuHisValSerMetPheGlnValValGlnGluGlnSer                              160165170                                                                     AACAGGGAGTCTGACTTGTTCTTTTTGGATCTTCAGACGCTCCGAGCT1056                          AsnArgGluSerAspLeuPhePheLeuAspLeuGlnThrLeuArgAla                              175180185                                                                     GGAGACGAGGGCTGGCTGGTGCTGGATGTCACAGCAGCCAGTGACTGC1104                          GlyAspGluGlyTrpLeuValLeuAspValThrAlaAlaSerAspCys                              190195200205                                                                  TGGTTGCTGAAGCGTCACAAGGACCTGGGACTCCGCCTCTATGTGGAG1152                          TrpLeuLeuLysArgHisLysAspLeuGlyLeuArgLeuTyrValGlu                              210215220                                                                     ACTGAGGACGGGCACAGCGTGGATCCTGGCCTGGCCGGCCTGCTGGGT1200                          ThrGluAspGlyHisSerValAspProGlyLeuAlaGlyLeuLeuGly                              225230235                                                                     CAACGGGCCCCACGCTCCCAACAGCCTTTCGTGGTCACTTTCTTCAGG1248                          GlnArgAlaProArgSerGlnGlnProPheValValThrPhePheArg                              240245250                                                                     GCCAGTCCGAGTCCCATCCGCACCCCTCGGGCAGTGAGGCCACTGAGG1296                          AlaSerProSerProIleArgThrProArgAlaValArgProLeuArg                              255260265                                                                     AGGAGGCAGCCGAAGAAAAGCAACGAGCTGCCGCAGGCCAACCGACTC1344                          ArgArgGlnProLysLysSerAsnGluLeuProGlnAlaAsnArgLeu                              270275280285                                                                  CCAGGGATCTTTGATGACGTCCACGGCTCCCACGGCCGGCAGGTCTGC1392                          ProGlyIlePheAspAspValHisGlySerHisGlyArgGlnValCys                              290295300                                                                     CGTCGGCACGAGCTCTACGTCAGCTTCCAGGACCTCGGCTGGCTGGAC1440                          ArgArgHisGluLeuTyrValSerPheGlnAspLeuGlyTrpLeuAsp                              305310315                                                                     TGGGTCATCGCTCCCCAAGGCTACTCGGCCTATTACTGTGAGGGGGAG1488                          TrpValIleAlaProGlnGlyTyrSerAlaTyrTyrCysGluGlyGlu                              320325330                                                                     TGCTCCTTCCCACTGGACTCCTGCATGAATGCCACCAACCACGCCATC1536                          CysSerPheProLeuAspSerCysMetAsnAlaThrAsnHisAlaIle                              335340345                                                                     CTGCAGTCCCTGGTGCACCTGATGAAGCCAAACGCAGTCCCCAAGGCG1584                          LeuGlnSerLeuValHisLeuMetLysProAsnAlaValProLysAla                              350355360365                                                                  TGCTGTGCACCCACCAAGCTGAGCGCCACCTCTGTGCTCTACTATGAC1632                          CysCysAlaProThrLysLeuSerAlaThrSerValLeuTyrTyrAsp                              370375380                                                                     AGCAGCAACAACGTCATCCTGCGCAAAGCCCGCAACATGGTGGTCAAG1680                          SerSerAsnAsnValIleLeuArgLysAlaArgAsnMetValValLys                              385390395                                                                     GCCTGCGGCTGCCACTGAGTCAGCCCGCCCAGCCCTACTGCAG1723                               AlaCysGlyCysHis                                                               400                                                                           (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 402 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetThrAlaLeuProGlyProLeuTrpLeuLeuGlyLeuAlaLeuCys                              151015                                                                        AlaLeuGlyGlyGlyGlyProGlyLeuArgProProProGlyCysPro                              202530                                                                        GlnArgArgLeuGlyAlaArgGluArgArgAspValGlnArgGluIle                              354045                                                                        LeuAlaValLeuGlyLeuProGlyArgProArgProArgAlaProPro                              505560                                                                        AlaAlaSerArgLeuProAlaSerAlaProLeuPheMetLeuAspLeu                              65707580                                                                      TyrHisAlaMetAlaGlyAspAspAspGluAspGlyAlaProAlaGlu                              859095                                                                        ArgArgLeuGlyArgAlaAspLeuValMetSerPheValAsnMetVal                              100105110                                                                     GluArgAspArgAlaLeuGlyHisGlnGluProHisTrpLysGluPhe                              115120125                                                                     ArgPheAspLeuThrGlnIleProAlaGlyGluAlaValThrAlaAla                              130135140                                                                     GluPheArgIleTyrLysValProSerIleHisLeuLeuAsnArgThr                              145150155160                                                                  LeuHisValSerMetPheGlnValValGlnGluGlnSerAsnArgGlu                              165170175                                                                     SerAspLeuPhePheLeuAspLeuGlnThrLeuArgAlaGlyAspGlu                              180185190                                                                     GlyTrpLeuValLeuAspValThrAlaAlaSerAspCysTrpLeuLeu                              195200205                                                                     LysArgHisLysAspLeuGlyLeuArgLeuTyrValGluThrGluAsp                              210215220                                                                     GlyHisSerValAspProGlyLeuAlaGlyLeuLeuGlyGlnArgAla                              225230235240                                                                  ProArgSerGlnGlnProPheValValThrPhePheArgAlaSerPro                              245250255                                                                     SerProIleArgThrProArgAlaValArgProLeuArgArgArgGln                              260265270                                                                     ProLysLysSerAsnGluLeuProGlnAlaAsnArgLeuProGlyIle                              275280285                                                                     PheAspAspValHisGlySerHisGlyArgGlnValCysArgArgHis                              290295300                                                                     GluLeuTyrValSerPheGlnAspLeuGlyTrpLeuAspTrpValIle                              305310315320                                                                  AlaProGlnGlyTyrSerAlaTyrTyrCysGluGlyGluCysSerPhe                              325330335                                                                     ProLeuAspSerCysMetAsnAlaThrAsnHisAlaIleLeuGlnSer                              340345350                                                                     LeuValHisLeuMetLysProAsnAlaValProLysAlaCysCysAla                              355360365                                                                     ProThrLysLeuSerAlaThrSerValLeuTyrTyrAspSerSerAsn                              370375380                                                                     AsnValIleLeuArgLysAlaArgAsnMetValValLysAlaCysGly                              385390395400                                                                  CysHis                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1926 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: cDNA                                                      (ix) FEATURE:                                                                 (A) NAME/KEY: CDS                                                             (B) LOCATION: 93..1289                                                        (D) OTHER INFORMATION: /function="OSTEOGENIC PROTEIN"                         /product= "mOP2-PP"                                                           /note= "mOP2 cDNA"                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       GCCAGGCACAGGTGCGCCGTCTGGTCCTCCCCGTCTGGCGTCAGCCGAGCCCGACCAGCT60                ACCAGTGGATGCGCGCCGGCTGAAAGTCCGAGATGGCTATGCGTCCCGGGCCA113                      MetAlaMetArgProGlyPro                                                         15                                                                            CTCTGGCTATTGGGCCTTGCTCTGTGCGCGCTGGGAGGCGGCCACGGT161                           LeuTrpLeuLeuGlyLeuAlaLeuCysAlaLeuGlyGlyGlyHisGly                              101520                                                                        CCGCGTCCCCCGCACACCTGTCCCCAGCGTCGCCTGGGAGCGCGCGAG209                           ProArgProProHisThrCysProGlnArgArgLeuGlyAlaArgGlu                              253035                                                                        CGCCGCGACATGCAGCGTGAAATCCTGGCGGTGCTCGGGCTACCGGGA257                           ArgArgAspMetGlnArgGluIleLeuAlaValLeuGlyLeuProGly                              40455055                                                                      CGGCCCCGACCCCGTGCACAACCCGCCGCTGCCCGGCAGCCAGCGTCC305                           ArgProArgProArgAlaGlnProAlaAlaAlaArgGlnProAlaSer                              606570                                                                        GCGCCCCTCTTCATGTTGGACCTATACCACGCCATGACCGATGACGAC353                           AlaProLeuPheMetLeuAspLeuTyrHisAlaMetThrAspAspAsp                              758085                                                                        GACGGCGGGCCACCACAGGCTCACTTAGGCCGTGCCGACCTGGTCATG401                           AspGlyGlyProProGlnAlaHisLeuGlyArgAlaAspLeuValMet                              9095100                                                                       AGCTTCGTCAACATGGTGGAACGCGACCGTACCCTGGGCTACCAGGAG449                           SerPheValAsnMetValGluArgAspArgThrLeuGlyTyrGlnGlu                              105110115                                                                     CCACACTGGAAGGAATTCCACTTTGACCTAACCCAGATCCCTGCTGGG497                           ProHisTrpLysGluPheHisPheAspLeuThrGlnIleProAlaGly                              120125130135                                                                  GAGGCTGTCACAGCTGCTGAGTTCCGGATCTACAAAGAACCCAGCACC545                           GluAlaValThrAlaAlaGluPheArgIleTyrLysGluProSerThr                              140145150                                                                     CACCCGCTCAACACAACCCTCCACATCAGCATGTTCGAAGTGGTCCAA593                           HisProLeuAsnThrThrLeuHisIleSerMetPheGluValValGln                              155160165                                                                     GAGCACTCCAACAGGGAGTCTGACTTGTTCTTTTTGGATCTTCAGACG641                           GluHisSerAsnArgGluSerAspLeuPhePheLeuAspLeuGlnThr                              170175180                                                                     CTCCGATCTGGGGACGAGGGCTGGCTGGTGCTGGACATCACAGCAGCC689                           LeuArgSerGlyAspGluGlyTrpLeuValLeuAspIleThrAlaAla                              185190195                                                                     AGTGACCGATGGCTGCTGAACCATCACAAGGACCTGGGACTCCGCCTC737                           SerAspArgTrpLeuLeuAsnHisHisLysAspLeuGlyLeuArgLeu                              200205210215                                                                  TATGTGGAAACCGCGGATGGGCACAGCATGGATCCTGGCCTGGCTGGT785                           TyrValGluThrAlaAspGlyHisSerMetAspProGlyLeuAlaGly                              220225230                                                                     CTGCTTGGACGACAAGCACCACGCTCCAGACAGCCTTTCATGGTAACC833                           LeuLeuGlyArgGlnAlaProArgSerArgGlnProPheMetValThr                              235240245                                                                     TTCTTCAGGGCCAGCCAGAGTCCTGTGCGGGCCCCTCGGGCAGCGAGA881                           PhePheArgAlaSerGlnSerProValArgAlaProArgAlaAlaArg                              250255260                                                                     CCACTGAAGAGGAGGCAGCCAAAGAAAACGAACGAGCTTCCGCACCCC929                           ProLeuLysArgArgGlnProLysLysThrAsnGluLeuProHisPro                              265270275                                                                     AACAAACTCCCAGGGATCTTTGATGATGGCCACGGTTCCCGCGGCAGA977                           AsnLysLeuProGlyIlePheAspAspGlyHisGlySerArgGlyArg                              280285290295                                                                  GAGGTTTGCCGCAGGCATGAGCTCTACGTCAGCTTCCGTGACCTTGGC1025                          GluValCysArgArgHisGluLeuTyrValSerPheArgAspLeuGly                              300305310                                                                     TGGCTGGACTGGGTCATCGCCCCCCAGGGCTACTCTGCCTATTACTGT1073                          TrpLeuAspTrpValIleAlaProGlnGlyTyrSerAlaTyrTyrCys                              315320325                                                                     GAGGGGGAGTGTGCTTTCCCACTGGACTCCTGTATGAACGCCACCAAC1121                          GluGlyGluCysAlaPheProLeuAspSerCysMetAsnAlaThrAsn                              330335340                                                                     CATGCCATCTTGCAGTCTCTGGTGCACCTGATGAAGCCAGATGTTGTC1169                          HisAlaIleLeuGlnSerLeuValHisLeuMetLysProAspValVal                              345350355                                                                     CCCAAGGCATGCTGTGCACCCACCAAACTGAGTGCCACCTCTGTGCTG1217                          ProLysAlaCysCysAlaProThrLysLeuSerAlaThrSerValLeu                              360365370375                                                                  TACTATGACAGCAGCAACAATGTCATCCTGCGTAAACACCGTAACATG1265                          TyrTyrAspSerSerAsnAsnValIleLeuArgLysHisArgAsnMet                              380385390                                                                     GTGGTCAAGGCCTGTGGCTGCCACTGAGGCCCCGCCCAGCATCCTGCTTCTACT1319                    ValValLysAlaCysGlyCysHis                                                      395                                                                           ACCTTACCATCTGGCCGGGCCCCTCTCCAGAGGCAGAAACCCTTCTATGTTATCATAGCT1379              CAGACAGGGGCAATGGGAGGCCCTTCACTTCCCCTGGCCACTTCCTGCTAAAATTCTGGT1439              CTTTCCCAGTTCCTCTGTCCTTCATGGGGTTTCGGGGCTATCACCCCGCCCTCTCCATCC1499              TCCTACCCCAAGCATAGACTGAATGCACACAGCATCCCAGAGCTATGCTAACTGAGAGGT1559              CTGGGGTCAGCACTGAAGGCCCACATGAGGAAGACTGATCCTTGGCCATCCTCAGCCCAC1619              AATGGCAAATTCTGGATGGTCTAAGAAGGCCCTGGAATTCTAAACTAGATGATCTGGGCT1679              CTCTGCACCATTCATTGTGGCAGTTGGGACATTTTTAGGTATAACAGACACATACACTTA1739              GATCAATGCATCGCTGTACTCCTTGAAATCAGAGCTAGCTTGTTAGAAAAAGAATCAGAG1799              CCAGGTATAGCGGTGCATGTCATTAATCCCAGCGCTAAAGAGACAGAGACAGGAGAATCT1859              CTGTGAGTTCAAGGCCACATAGAAAGAGCCTGTCTCGGGAGCAGGAAAAAAAAAAAAAAC1919              GGAATTC1926                                                                   (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 399 amino acids                                                   (B) TYPE: amino acid                                                          (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      MetAlaMetArgProGlyProLeuTrpLeuLeuGlyLeuAlaLeuCys                              151015                                                                        AlaLeuGlyGlyGlyHisGlyProArgProProHisThrCysProGln                              202530                                                                        ArgArgLeuGlyAlaArgGluArgArgAspMetGlnArgGluIleLeu                              354045                                                                        AlaValLeuGlyLeuProGlyArgProArgProArgAlaGlnProAla                              505560                                                                        AlaAlaArgGlnProAlaSerAlaProLeuPheMetLeuAspLeuTyr                              65707580                                                                      HisAlaMetThrAspAspAspAspGlyGlyProProGlnAlaHisLeu                              859095                                                                        GlyArgAlaAspLeuValMetSerPheValAsnMetValGluArgAsp                              100105110                                                                     ArgThrLeuGlyTyrGlnGluProHisTrpLysGluPheHisPheAsp                              115120125                                                                     LeuThrGlnIleProAlaGlyGluAlaValThrAlaAlaGluPheArg                              130135140                                                                     IleTyrLysGluProSerThrHisProLeuAsnThrThrLeuHisIle                              145150155160                                                                  SerMetPheGluValValGlnGluHisSerAsnArgGluSerAspLeu                              165170175                                                                     PhePheLeuAspLeuGlnThrLeuArgSerGlyAspGluGlyTrpLeu                              180185190                                                                     ValLeuAspIleThrAlaAlaSerAspArgTrpLeuLeuAsnHisHis                              195200205                                                                     LysAspLeuGlyLeuArgLeuTyrValGluThrAlaAspGlyHisSer                              210215220                                                                     MetAspProGlyLeuAlaGlyLeuLeuGlyArgGlnAlaProArgSer                              225230235240                                                                  ArgGlnProPheMetValThrPhePheArgAlaSerGlnSerProVal                              245250255                                                                     ArgAlaProArgAlaAlaArgProLeuLysArgArgGlnProLysLys                              260265270                                                                     ThrAsnGluLeuProHisProAsnLysLeuProGlyIlePheAspAsp                              275280285                                                                     GlyHisGlySerArgGlyArgGluValCysArgArgHisGluLeuTyr                              290295300                                                                     ValSerPheArgAspLeuGlyTrpLeuAspTrpValIleAlaProGln                              305310315320                                                                  GlyTyrSerAlaTyrTyrCysGluGlyGluCysAlaPheProLeuAsp                              325330335                                                                     SerCysMetAsnAlaThrAsnHisAlaIleLeuGlnSerLeuValHis                              340345350                                                                     LeuMetLysProAspValValProLysAlaCysCysAlaProThrLys                              355360365                                                                     LeuSerAlaThrSerValLeuTyrTyrAspSerSerAsnAsnValIle                              370375380                                                                     LeuArgLysHisArgAsnMetValValLysAlaCysGlyCysHis                                 385390395                                                                     (2) INFORMATION FOR SEQ ID NO:11:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 884 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..884                                                          (D) OTHER INFORMATION: /note= "hOP-2 genomic sequence"                        (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1..837                                                          (D) OTHER INFORMATION: /note= "EXON ONE"                                      (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 884                                                             (D) OTHER INFORMATION: /note= "A GAP OCCURS BETWEEN                           POSTIONS 884 IN THIS SEQUENCE AND POSITION 1 IN                               SEQ ID NO 14"                                                                 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:                                      GGAATTCCGGCCACAGTGGCGCCGGCAGAGCAGGAGTGGCTGGAGGAGCTGTGGTTGGAG60                CAGGAGGTGGCACGGCAGGGCTGGAGGGCTCCCTATGAGTGGCGGAGACGGCCCAGGAGG120               CGCTGGAGCAACAGCTCCCACACCGCACCAAGCGGTGGCTGCAGGAGCTCGCCCATCGCC180               CCTGCGCTGCTCGGACCGCGGCCACAGCCGGACTGGCGGGTACGGCGGCGACAGACGGAT240               TGGCCGAGAGTCCCAGTCCGCAGAGTAGCCCCGGCCTCGAGGCGGTGGCGTCCGCGTCCT300               CTCGTCCAGGAGCCAGGACAGGTGTCGCGCGGCGGGCCGTCCAGGGACCGCGCTGAGGCC360               GCGGTCGCCCGTCCCGCCCCGCCCCGCCGCCCGCCGCCCGCCGAGCCCAGCCTCCTTGCC420               GTCGGGGCGTCCCCAGGCCCTGGGTCGGCCGCGGAGCCGATGCGCGCCCGCTGAGCGCCC480               CAGCTGAGCGCCCCCGGCCTGCCATGACCGCGCTCCCCGGCCCGCTCTGGCTCCTGGGCC540               TGGCGCTATGCGCGCTGGGCGGGGGCGGCCCCGGCCTGCGACCCCCGCCCGGCTGTCCCC600               AGCGACGTCTGGGCGCGCGCGAGCGCCGGGACGTGCAGCGCGAGATCCTGGCGGTGCTCG660               GGCTGCCTGGGCGGCCCCGGCCCCGCGCGCCACCCGCCGCCTCCCGGCTGCCCGCGTCCG720               CGCCGCTCTTCATGCTGGACCTGTACCACGCCATGGCCGGCGACGACGACGAGGACGGCG780               CGCCCGCGGAGCGGCGCCTGGGCCGCGCCGACCTGGTCATGAGCTTCGTTAACATGGGTG840               AGTGCGGCGCCCGCGCGGGGACCCTCGGAGTAAACTGGCTGCAG884                               (2) INFORMATION FOR SEQ ID NO:12:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 97 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..97                                                           (D) OTHER INFORMATION: /label=Generic-Seq-7                                   /note= "wherein each Xaa is independently selected from                       a group of one or more specified amino acids as defined                       in the specification."                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:                                      LeuXaaXaaXaaPheXaaXaaXaaGlyTrpXaaXaaXaaXaaXaaXaa                              151015                                                                        ProXaaXaaXaaXaaAlaXaaTyrCysXaaGlyXaaCysXaaXaaPro                              202530                                                                        XaaXaaXaaXaaXaaXaaXaaXaaAsnHisAlaXaaXaaXaaXaaXaa                              354045                                                                        XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaCysCysXaaPro                              505560                                                                        XaaXaaXaaXaaXaaXaaXaaXaaLeuXaaXaaXaaXaaXaaXaaXaa                              65707580                                                                      ValXaaLeuXaaXaaXaaXaaXaaMetXaaValXaaXaaCysXaaCys                              859095                                                                        Xaa                                                                           (2) INFORMATION FOR SEQ ID NO:13:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 102 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (ix) FEATURE:                                                                 (A) NAME/KEY: Protein                                                         (B) LOCATION: 1..102                                                          (D) OTHER INFORMATION: /label=Generic-Seq-8                                   /note= "wherein each Xaa is independently selected from                       a group of one or more specified amino acids as defined                       in the specification."                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:                                      CysXaaXaaXaaXaaLeuXaaXaaXaaPheXaaXaaXaaGlyTrpXaa                              151015                                                                        XaaXaaXaaXaaXaaProXaaXaaXaaXaaAlaXaaTyrCysXaaGly                              202530                                                                        XaaCysXaaXaaProXaaXaaXaaXaaXaaXaaXaaXaaAsnHisAla                              354045                                                                        XaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaaXaa                              505560                                                                        XaaCysCysXaaProXaaXaaXaaXaaXaaXaaXaaXaaLeuXaaXaa                              65707580                                                                      XaaXaaXaaXaaXaaValXaaLeuXaaXaaXaaXaaXaaMetXaaVal                              859095                                                                        XaaXaaCysXaaCysXaa                                                            100                                                                           (2) INFORMATION FOR SEQ ID NO:14:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 950 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "A GAP OCCURS BETWEEN                           POSTION 884 IN SEQ ID NO: 11 AND POSITION 1 IN THIS                           SEQUENCE"                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 204..393                                                        (D) OTHER INFORMATION: /note= "EXON TWO"                                      (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 466..930                                                        (D) OTHER INFORMATION: /note= "EXON THREE"                                    (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..950                                                          (D) OTHER INFORMATION: /note= "hOP-2 genomic sequence"                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:                                      CTGCAGGGCCTCTTCTGGCTCTACACCCCGGGACCAAGCCTGGAACAAACGTTTGCACTA60                AATGAAGCCGGCCCCACCCAGGCCTCCCTGGGTCCGCTCCACCTTGAGTGGTGGGTGGCT120               GGGGGCGGTGGCTCACACCAGCTCTGCCCCCTCCAGAGCCCGAGCCATTCTGAGTGCCAG180               CCCAGCGCTGCTTTGTCTTCTAGTGGAGCGAGACCGTGCCCTGGGCCACCAGGAGCCCCA240               TTGGAAGGAGTTCCGCTTTGACCTGACCCAGATCCCGGCTGGGGAGGCGGTCACAGCTGC300               GGAGTTCCGGATTTACAAGGTGCCCAGCATCCACCTGCTCAACAGGACCCTCCACGTCAG360               CATGTTCCAGGTGGTCCAGGAGCAGTCCAACAGGTGCCTTCCCCTTGGCCCGGGTGCCCA420               CCTAACCCCCCACCTCACAGTCTCATGGTCAAGGCAGCCCAGCAGGGAGTCGTGGTGGGT480               GAAAGAGAGCCTCAAAGATGGGAAGGATGCTTGGCCCGAGGCCCTGCACTGTGGGAAGAG540               CCCCAGTGACAATCCTGACTTCAAGTCCCTGCCCTCCATCCTGCTGTGGGGACTTGGACA600               TGGTCACTGAGACTCAGTTTCCCCATGTGTACACCTCTGTGGGCTGAGGCAATGAGATGA660               GGCTCAGAAGGGCGCAGCCAGAGTCAGGTGGGAGACGCTCCGGTGACAGCCCCCAGCGGG720               CCCTGGAGACACGGAGGCAGCTGTGCCGGCCGCCGGTTAATTGTTCTTTCATGTCCACAG780               GGGAGTCTGACTTGTTCTTTTTGGATCTTCAGACGCTCCGAGCTGGAGACGAGGGCTGGC840               TGGTGCTGGATGTCACAGCAGCCAGTGACTGCTGGTTGCTGAAGCGTCACAAGGACCTGG900               GACTCCGCCTCTATGTGGAGACTGAGGACGGTGAGGCTGGGGCTCTGCAG950                         (2) INFORMATION FOR SEQ ID NO:15:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 4584 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: DNA (genomic)                                             (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1..4584                                                         (D) OTHER INFORMATION: /note= "hOP-2 genomic sequence"                        (ix) FEATURE:                                                                 (A) NAME/KEY: misc.sub.-- feature                                             (B) LOCATION: 1                                                               (D) OTHER INFORMATION: /note= "A GAP OCCURS BETWEEN                           POSITION 950 IN SEQ ID NO: 14 AND POSITION 1 IN THIS                          SEQUENCE"                                                                     (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 49..243                                                         (D) OTHER INFORMATION: /note= "EXON FOUR"                                     (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1064..1147                                                      (D) OTHER INFORMATION: /note= "EXON FIVE"                                     (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 1673..1783                                                      (D) OTHER INFORMATION: /note= "EXON SIX"                                      (ix) FEATURE:                                                                 (A) NAME/KEY: exon                                                            (B) LOCATION: 4282..4527                                                      (D) OTHER INFORMATION: /note= "EXON SEVEN"                                    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:                                      CTGCAGAGCCACTGCCCGTGAGTGACCCCTCTCTCCTTTCTGTCTCAGGGCACAGCGTGG60                ATCCTGGCCTGGCCGGCCTGCTGGGTCAACGGGCCCCACGCTCCCAACAGCCTTTCGTGG120               TCACTTTCTTCAGGGCCAGTCCGAGTCCCATCCGCACCCCTCGGGCAGTGAGGCCACTGA180               GGAGGAGGCAGCCGAAGAAAAGCAACGAGCTGCCGCAGGCCAACCGACTCCCAGGGATCT240               TTGGTGAGGGTCGGGCAGGCTGGGGCGAGGCTGTGGCTGTCTGGCTGAGAGAGGCAGGGC300               GAGAACCAAGTGGTGGCCCAGAGCCCAGAGCCTCAGGCTAGGTCGGTTCAAGCTGACGGC360               CACTCTCCAGCCACCTTTCCTGACACCATCTTGGCCCTGATGCACCCTGGTGACCGGCAC420               TCCGAGGCCTGTCCTGGCTGTCCCTGCTGCCAGAAGTCTCCCTCTCTCCCCCTGGCTCCT480               CCGGGTCTTTCTCAGGAGCCTCCTTCAGAATCAGCTGCCCCTTCCCTGGGAGCCGCAGCC540               CCTCATGACCTGCGGTTGTGCCTGGGCACCTGTGGATCCTCGGTTGCTTATGCGATTTTC600               TCCCCAACTGGCCAAGCTTCAGGATCAGGGACAGGCCTGACCCAACCCCGTGCCCTCCTT660               CCCAGGGAGTCGGCCCTTGACTGGCCTGGTCGTGAGCCACTTGAACCTCGGGAATGGGTG720               TGGCAGGAGAGGGTGGGCTGGAGTCACAGGGGTCTCCAGAGAGGAGGAGGCACAGGATGG780               CCGAGGGTCCTGCTGGGCTGTTTACTGGAGCATAAAGATGCTCATAGGCTGAAGGACAGG840               GGAGGACTGGGCACAGTGTCACTCTAGCCATTGGGAGCCATGGCAGGCTTCTGAGCTGGG900               TCATGGTACAAGCAGAGTTCCAGGGATGGGCTTTATGAGCCAAATGGTTTCCTGTCATTC960               ATTTATTTGACAAATGTGCTCATCAGGGCATCCCCCACCCTGGTACCCCATAGTAGCTGC1020              ACACAGCAGGAACCCCAGAAAAGACCTTGCCCCTTCTGTCCCTGCAGATGACGTCCACGG1080              CTCCCACGGCCGGCAGGTCTGCCGTCGGCACGAGCTCTACGTCAGCTTCCAGGACCTCGG1140              CTGGCTGGTAATTGCTGACTCTCCTTGTTTCTGAAATGACAATCACCACCTGTAGATCAG1200              AAGTGAATCTGCAGGGAGGACATAGAATCATGGTGACTTCAATTTTCTTATGTATTTTTT1260              TCTTCTGTGTTTTCCAAGTTTTCTAAAGTGAGAATATGGTGAGAAAGGGTTTTGTTGTTG1320              TTGTTGTGTTTTTTGTTTTTTTTTAAAAACCCATGAAAATGAAGACTGAATCAACCAACT1380              AAGCTGTCAGCATTGCCGCAGGGTAACTGAGACCTCCCTGCATTGGCTACGACTGCAGCT1440              CTGGGAGGTGTGGGCAGGGGAGGGCCGGCTGGGGAGGGCCGGCTGGGGAGGGGACACAAA1500              GTGAAGATGGGGGTTGTTGGGCCTGAGCTCCTGCCCAGCCTTTTCCGCCGGGGTTCCTGG1560              GTGGATTCAAGCCTCTTGGGGGAGACGCGCTGCAGGGCTGGAGGATGGGCTTTGGGCCCT1620              GAGGCTCAGGGAGGAGCACATGGATGGGACTCACCTTCTCCCTTGCCCCCAGGACTGGGT1680              CATCGCTCCCCAAGGCTACTCGGCCTATTACTGTGAGGGGGAGTGCTCCTTCCCACTGGA1740              CTCCTGCATGAATGCCACCAACCACGCCATCCTGCAGTCCCTGGTCGATACCGTCGCCCA1800              TCCTGCCCAGCCCCCTGGTGGAGGCCCTGCAGAGAGGGGTCTGGTCCAGCCAGCCGGGAG1860              GCAGTGAGGCCACCTGCTCCATGTCTCGGGGCTTTGTCTGCACAGAGTCAGTAACGTCGC1920              TAACTTCCCACAGCTCTGCAGGAACTGGTCCTCATACAGCCACACTACTACACATAGACC1980              CACACCCAAACACGGACACACGTGAACAGTCGCGTATCATGCCTGTTCTATGCACTGAAC2040              AAACTCCTGTGGGACACTTACACACCTGCGTGCGGCGCTCAGAGGCACAGCACATGAAAC2100              AGATGTGTACACTGTGTGGGGGCTGTGTGATCTTAACACACGGGCCCCCGAGTACGCTGG2160              CAAGTCTGACCGCCCGTGATATGTGCGCACAGTGTGTGGGGTGTGCGTGTGCATCACCCA2220              CCTGTGCCGCACCACAGGTAGGAAGCTTCTAGATGGTGTGGCTCTCAACCTTTTGGCTTT2280              TTCCCGCAGTTTCTCTCTTGGCTGTCTGTGTTTTCTCTGGATCCCCTGGCTTTTGATGCC2340              GTTGGTGTCTGGGGCAACCTTAAAGGACAAAAGCAGGCTTCTGATGGGATCACTGGTGCT2400              GCTCACCACTGAGTGCTCGTGTGTTTGCGGATTCTGGCACCGAGGCTTCCTTCTAGAAGT2460              TTTTACCTAGAATCCCAGTTCCTGGTATTGCACAGCCTTATGTTTTCCTCTTAGGAGGTT2520              CAACGGTGATGCCTTGATCAGGCGCAGTGGCTCACCCTGTAATCGCAGCACACGAGCCCA2580              GAAGTTCAAGACAAGCCTGAGCAACACAGCAAAACCCTGTCTCTAAAATAAAAATTAAAA2640              CACACACACACACACACACACACACACACACACGTGCGCACACAATGCCTTGGTGTGAGA2700              GGAAAGAAATTACCAAAAGCTGCTCTGAGCCTATGATAATACTTCCTTTCTGGGCAGTCA2760              AATGGTGTTTGCTGGACACCCTGGAGCCATCTCCTTGGAAAGGCCCAGGGGTGATGAGGA2820              GCTCCGTCGGGGTGGCCTGGCCAGCACCTTTATGCCGTGTGGTTCTCACAGCTGCATGTG2880              TGGGAGGTACATGGGAAGGTGACTGCACCTGCGCTCCTGGACTCCATCTCCTCTGCCCTT2940              GCCCCTGCCCCTCACGTGCAACTAGAGTGAGTGCTCACAGCCTACAGGGCAGCAAACAGG3000              CACTGTGCTCTAGGGGAGGCTGTCGGTGGGCACAGAAGCAAACCAACCGTGGAGTTGACA3060              CCTCCTGTGAGGAAGAGCAGACGAGCCGTGCCGTCAGTGGAGTGAGACTGGGCCCAGCTC3120              TCCACACAAGGAGGGGCACGTCAGCAGCTGGAGGAGGAATGTTCCAGAAGGAGCAAGTGC3180              AAGGCCCTAAGACAGGAGCAGGCTGGCCCTAAGTTCAGGGCAGGGGAGGAGAGGGGCTGG3240              GTGCAGTGAAGGGGAGGAGAGTGGAGGGAGGTGATCCGGGGTGATAGGCCAGCTCCCGTA3300              GCCTGGGTTCCCTGGGAAGAGGGTGGATTTTATTCCAAGCAACCCCAGAGGCTGTCAGAG3360              GTCTTCAGCAAAGAGTGTCCTTGGTCTGCGTCACCCTCCAGAAGGACCTTTCTGGCTTGG3420              GGAGGTCGCGGGAGTGGAAGGCAGAGGAGCAGGGGATGAGTGAGGGCTGCTGTGGTCACC3480              TGGCAGGTGATGGCAGCTCGACTGGGCAGGTGGTCCGAGGCAGCACGGAGGTGGAGGTTG3540              AGCCAGGGGCTGCTCTCAGGGAAGGGAGGAGGCGAAAGGAGTCATCCAGGAGGCCTCCCA3600              GGCGGGAGCTATGATGTCAGGGCGGGAGGAATTCTATGTTCCACTGAGGCCTCATTAGAC3660              CCCCAAGTGCAGAAGTGGGAAGGGGAGCAGGATCCGCAAGTCTGGAGTTCAGAAGAGAGG3720              TCCAAGCTGAGCCAGGGGAGTGGAGAGGTGCGGGCCAATGCAGGGCCTTGAAGTGCTGAG3780              GGCGGATCGAGTCCTCTGGGAGAAGGAGCAGCACAGGAGAGGGGGCGAGGCTGGCTCCCA3840              GAGCCTGGGGAGGGAGGCAGGTGTGGGGAGGCAGAGCTTGGGGGGGTCTGAAGGGCTATA3900              AGAAGACAGTGGTCCTTCCAGGTTCCCCCTTGGACCTCACTAAGGGCACAAACCTGGCCA3960              TGAGGTTCTCCTTCCCATTATCCCCAGGAGGAAGTCTGAGCCCTTGGCCTGGGACTCGAG4020              GCCCCTCATTAGTGCCCTGCCCACCTGCCCCACACCCTGGGGCTGCCATGTATCCCTCCC4080              TGGGCACTGTGGGCACCACAGCTCCCGCTCCCAGAGCTCTCAGGGCTGCTCTTATTCCTG4140              TTAATAATTCTTATTATTGTGCTGCTCCCATGTGGCTTGGAGATGGCCAGGGCAGGGAGC4200              AGGTGGAGCTGGGGCGGGCTAGGTGGGTCCTCAGAGGAGGCCACTGGCTCATGCCCCTGC4260              CTGTGCTCCCTTCCTGGCCAGGTGCACCTGATGAAGCCAAACGCAGTCCCCAAGGCGTGC4320              TGTGCACCCACCAAGCTGAGCGCCACCTCTGTGCTCTACTATGACAGCAGCAACAACGTC4380              ATCCTGCGCAAGCACCGCAACATGGTGGTCAAGGCCTGCGGCTGCCACTGAGTCAGCCCG4440              CCCAGCCCTACTGCAGCCACCCTTCTCATCTGGATCGGGCCCTGCAGAGGCAGAAAACCC4500              TTAAATGCTGTCACAGCTCAAGCAGGAGTGTCAGGGGCCCTCACTCTCTGTGCCTACTTC4560              CTGTCAGGCTTCTGGTCCTTTCTC4584                                                  __________________________________________________________________________

What is claimed is:
 1. An in vitro or ex vivo composition comprising aprogenitor cell population isolated from a renewable or conditionallyrenewable tissue and at least a fragment of OP-3, wherein said fragmentinduces endochondral bone formation in an in vivo assay.
 2. Thecomposition of claim 1, wherein said fragment comprises amino acids 303to 399 of SEQ ID NO: 1 or a conservative variant thereof.
 3. Thecomposition of claim 2 wherein said fragment has an amino acidsubstitution for the serine at position 315 or the cysteine at position338 in SEQ ID NO:
 1. 4. The composition of claim 2, wherein saidfragment comprises amino acids 298 to 399 of SEQ ID NO: 1 or aconservative variant thereof.
 5. The composition of claim 4, whereinsaid fragment comprises amino acids 264 to 399 of SEQ ID NO: 1 or aconservative variant thereof.
 6. The composition of claim 5, whereinsaid fragment comprises amino acids 261 to 399 of SEQ ID NO: 1 or aconservative variant thereof.
 7. The composition of claim 6, whereinsaid fragment comprises amino acids 18 to 399 of SEQ ID NO:1 or aconservative variant thereof.
 8. The composition of claim 7, whereinsaid fragment comprises amino acids 1 to 399 of SEQ ID NO: 1 or aconservative variant thereof.